RNA-nanostructured double robots and methods of use thereof

ABSTRACT

Described herein are immuno-stimulatory RNA nanostructures (which comprises a single-stranded RNA (ssRNA) molecule, wherein the ssRNA molecule forms at least one paranemic cohesion crossover), as well as compositions and methods of use thereof.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 National Stage Entry ofInternational Application No. PCT/US2019/013118, filed on Jan. 10, 2019,which claims the benefit of priority of U.S. Provisional Application No.62/615,806, filed on Jan. 10, 2018, the entire disclosures of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 15, 2020, isnamed G8118-01002_SL.txt and is 48,138 bytes in size.

FIELD

The present invention relates to compositions and methods for treatingpatients with cancer using immuno-stimulatory RNA nanostructures. Theinvention also includes methods of creating immuno-stimulatory RNAnanostructures, and compositions comprising said nanostructures.

BACKGROUND

Single stranded RNA (ssRNA) and double stranded RNA (dsRNA) can bedetected by pattern recognition receptors in mammalian cells andsynthetic ssRNA and dsRNA have been explored as immuno-stimulatingadjuvants (Alexopoulou, et al., 2001. Nature 413:732-738.). For example,polyinosinic:polycytidylic acid (polyIC), a synthetic analog of dsRNA,has been widely studied as an adjuvant in treating diseases such asupper respiratory tract infections and tumors, therefore, allowing it tobe explored as an adjuvant in flu and cancer vaccines. However,susceptibility of dsRNA to nuclease digestion tends to be a concernespecially when they are used in vivo.

TLR3-ligands have been used as adjuvants in cancer vaccination. PolyIChas been used in combination with tumor-specific antigens (TSAs) toinduce T-cell dependent responses against tumor cells. PolyIC was mixedwith TSAs such as a model antigen, ovalbumin (46 Kd) or with peptidesenwrapped within lipid. PolyIC rendered cross-presentation ofinternalized antigens for an induction of CD8+ cytotoxic T cellactivity.

Heat shock proteins (HSPs) with the molecular weights of approximately70 and 90 kDa have the capacity to stimulate antitumor immune responseseither as carriers for antigenic peptides. (Shevtsov M. and Multhoff G.Heat Shock Protein-Peptide and HSP-Based Immunotherapies for theTreatment of Cancer, 2016 Apr. 29; 7:171, Frontiers in Immunology, seeFIG. 15.) Heat Shock Protein-70 (HSP70) and derived peptides (alsoreferred to as TPP or TKD) function as chaperones. The chaperonepeptides can act as tumor-specific antigens and as immunogens. LinkingHSP70 to nanoparticlesllows for the capture of tumor cell lysates topresent antigens to dendritic cells (DCs). HSP70 protein and derivedpeptides can pre-activate NK cells for direct killing of HSP-70+ tumorcells. Dose-dependent and saturable enhancement was found at 0.2-2.0μg/ml for activation, and at >4 μg/ml no responses. HSP70 induced theproliferation of tumor cells, induced NK cell migration toward HSP70+tumor cells, the lysis of HSP70+ tumor cells by binding to granzymes andinducing apoptosis of target cells, and increased CD94 expression thatcan associate with NKG2A and bind to HSP70 to engage with tumor cells.HSP70 also increase dDC maturation and cross-presentation, increased Th1and CTL activity, and increased M1 activity.

HSP70/TKD moved to clinical trials (I & II), where one out of 12patients with brain tumor showed CR, who showed increased Th1 andreduced Treg, and where 7 out of 12 patients with HCV-HCC showedcomplete remission (CR) or stable disease (SD) after receivingHSP70-mRNA transfected to DC.

Accordingly, safe and effective molecular-cargo delivery nano-scaffoldsand methods are needed.

SUMMARY

This disclosure provides for RNA nanostructure robots and compositionscomprising the same for the treatment of a disease or disorder. In someaspects, the disease or disorder is cancer.

In certain aspects, the present invention provides a RNA nanostructure(also referred to herein as “RNA origami” or “OG-RNA” or “RNA-OG”)having the sequence of (R₃)_(n)-NR₁-L-NR₂-(R₄)_(m)—wherein:

-   -   NR₁ represents a first nano-robot comprising a single stranded        RNA (ssRNA) of about 1500 to 10,000 bases in length that        self-assembles into a first scaffold;    -   NR₂ represents a second nano-robot comprising a ssRNA, or a DNA        cage, of about 1500 to 10,000 bases in length that        self-assembles into a second scaffold;    -   L is a linker which operably links NR₁ to NR₂;    -   wherein R₃ and R₄ are independently selected from a pair of        fastener strands, an aptamer, a cargo molecule, a capture        strand, a targeting strand, or H;    -   n is an integer from 0 to 20; and    -   m is an integer from 0 to 20.

In some aspects, the ssRNA can comprise the sequence of:

-   -   (HD₁-LD₁-HD₂-LD₂)_(x)    -   wherein x is selected from 2 to 100, 2 to 500, 2 to 1000, 2 to        1500, 2 to 2000, 2 to 2500, 2 to 3000, or 2 to 3500, 2 to 4000,        2 to 4500, 2 to 5000, 2 to 5500, or 2 to 6000;    -   wherein HD₁ and HD₂ are each an RNA helical domain;    -   wherein LD₁ and LD₂ are each an RNA locking domain;    -   and further wherein the ssRNA sequence, when folded, exhibits at        least one paranemic cohesion crossover.

In certain aspects, the RNA nanostructure robot is an RNA nanostructuredouble robot or a double nanostructure comprising polynucleotides, whereNR₁ and NR₂ are assembled separately and joined by a linker L. In acertain aspect, the RNA nanostructure is comprised of two or moremotifs, wherein the first nano-robot comprises a first motif, and thesecond nano-robot comprises a second motif. In another aspect, thedouble nanostructure comprises an RNA nanorobot and a DNA cage. In theseand certain aspects, the RNA nanorobot and DNA cage are linked via alinker L. In some aspects, the first and second motifs can be separatelytranscribed as two separate polynucleotide chains, which are then linkedtogether through the linker L. In certain aspects, the linker L betweenNR₁ and NR₂ can be any group that can connect NR₁ and NR₂ to each other,as disclosed herein, provided that it does not interfere with thefunction of the NR₁ to NR₂, R₃ and/or R₄ moieties. In certain aspects,the linker L between NR₁ and NR₂ can be any group that can connect theNR₁ or NR₂ RNA nanostructure robots to each other, as disclosed herein,provided that it does not interfere with the function of the NR₁ to NR₂,R₃ and/or R₄ moieties, or the RNA nanostructure or DNA cage. In certainaspects, the linker L is selected from an oligonucleotide, ahybridization complex comprising two DNA or RNA sequences or portionsthereof, a DNA-RNA hybridization complex, a polymer, a peptide, an alkylchain, a polyethylene glycol (PEG) chain, a polypropylene glycol (PPG)chain, or combinations thereof. In some aspects, the linker L comprisesRNA ribonucleotides. In some aspects, the oligonucleotide is comprisedof DNA, RNA, modified DNA, modified RNA, or combinations thereof. Insome aspects, the linker L comprises deoxyribonucleotides. In someaspects, the linker L is a hybridization complex comprising two separateDNA or RNA strands, wherein a first DNA or RNA strand is part of thefirst nanorobot (NR₁), and a second DNA or RNA strand is part of thesecond nanorobot (NR₂). In certain aspects, portions of the two separateDNA or RNA chains can be hybridized to each other to form a linker. Insome aspects, the hybridization can be a direct hybridization between aportion of the first DNA or RNA chain which is complementary to aportion of the second DNA or RNA chain. In some aspects, thehybridization can be an indirect hybridization via a bridgeoligonucleotide wherein a portion of the sequence of one terminus of thebridge oligonucleotide is complementary to a portion of the sequence ofthe first DNA or RNA chain, and a sequence of the other terminus of thebridge oligonucleotide is complementary to a portion of the sequence ofthe second DNA or RNA chain, wherein hybridization occurs betweenportions of each of the first and second DNA or RNA chains and thebridging oligonucleotide. In some aspects, when NR₂ is a DNA cage, thelinker L can be an oligonucleotide connected to NR₂ which hybridizeswith a polyribonucleotide sequence connected to NR₁. In other aspects,the two separate DNA or RNA chains can be joined by a chemicalcross-link. The chemical cross-link can be a cross-link which covalentlybinds the two separate DNA or RNA chains to each other. The chemicalcross-link can be achieved through the incorporation of Psoralen intoone of the DNA or RNA chains, and upon photo-irradiation forms achemical bond to the other DNA or RNA chain. In some aspects, thechemical cross-link can be a binfunctional compound which reacts to amodified DNA or RNA in each of the separate DNA or RNA strands.

In some aspects, NR₂ is a DNA nanocage. As used herein, the term “DNAnanocage” refers to comprises a three dimensional body comprising aplurality of structural members comprising DNA, wherein internalsurfaces of the plurality of structural members form an inner cavity.The DNA can be M13 viral DNA. DNA nanocages can be those described inU.S. patent application Ser. No. 15/649,351, herein incorporated byreference in its entirety.

In certain aspects, the RNA nanostructure robot is a single chain, wherethe sequence NR₁-L-NR₂ is continuous. The NR₁-L-NR₂ sequence can betranscribed as a single chain (ssRNA) before functionalizing with R₃ andR₄. In some aspects, one or more of R₃ and/or R₄ is the same ordifferent. In some aspects, when R₃ and R₄ are different, there are morethan one species of R₃. In some aspects, when R₃ and R₄ are different,there are more than one species of R₄. In some aspects, there are fromone to 20 different types of species. In some aspects, when there aremore than one species of R₃, a first species of R₃ (“R_(3a)”) can be anaptamer, and a second species of R₃ (“R_(3b)”) is a peptide. In someaspects, when there are more than one species of R₄, a first species ofR₄ (“R_(4a)”) is an aptamer, and a second species of R₄ (“R_(4b)”) is apeptide.

In certain aspects, the present invention provides for a RNAnanostructure as described herein where the first scaffoldself-assembles into a rectangular shape.

In certain aspects, the present invention provides for a RNAnanostructure robot that further comprises a cargo molecule. In someaspects, R₄ is a cargo molecule and m is an integer from 1 to 20. Thecargo molecule can be an aptamer, protein, or drug molecule. In someaspects, the protein is an antigen. In some aspects, the cargo moleculeis operably linked to NR₂.

In some aspects, the present invention provides for a RNA nanostructurerobot that further comprises one or more fastener strands of DNA,wherein the one or more molecular fasteners are capable of fastening thefirst or second scaffold into an origami structure. In some aspects, R₃is a pair of fastener strands which comprise DNA, and the pair offasteners strands are capable of fastening the first or second scaffoldinto an origami structure, and n is an integer from 1 to 20. Each of thefastener strands of DNA can comprise a first and a second strand of DNA.The first and second strand of DNA can be selected from a sequence pairof the following oligonucleotides:

5′-FITC-labeled F50 and 3′-BHQ1-labeled Comp 15;FITC-F50-48 and Comp15-48-Q; FITC-F50-73 and Comp15-73-Q;FITC-F50-97 and Comp15-97-Q; FITC-F50-120 and Comp15-120-Q;FITC-F50-144 and, Comp15-144-Q; or FITC-F50-169 and Comp15-169-Q;wherein the aforementioned oligonucleotideshave the following sequences: 5′-FITC-labeled F50: (SEQ ID NO: 10)5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGT GAATTGCG-3′;3′-BHQ1-labeled Comp15: (SEQ ID NO: 11)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; FITC-F50-48: (SEQ ID NO: 12)5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGT GAATTGCG-3′;Comp15-48-Q: (SEQ ID NO: 13)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-73(SEQ ID NO: 14) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGGAAATCAAAA-3′; Comp15-73-Q: (SEQ ID NO: 15)5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-97:(SEQ ID NO: 16) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGAACAAACTAT-3′; Comp15-97-Q: (SEQ ID NO: 17)5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-120:(SEQ ID NO: 18) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTACAAACAGTT-3′; Comp15-120-Q: (SEQ ID NO: 19)5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-144:(SEQ ID NO: 20) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTTCCTATTATT-3;; Comp15-144-Q: (SEQ ID NO: 21)5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-BHQ1′-3′; FITC-F50-169:(SEQ ID NO: 22) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTCACCTGAAA-3′; Comp15-169-Q: (SEQ ID NO: 23)5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′.

F50 and Comp15; F50-48 and Comp15-48; F50-73 and Comp15-73;F50-97 and Comp15-97; F50-120 and Comp15-120;F50-144 and, Comp15-144; or F50-169 and Comp15-169;wherein the aforementioned oligonucleotideshave the following sequences: F50: (SEQ ID NO: 24)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGAATT GCG-3′; Comp15:(SEQ ID NO: 25) 5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; F50-48:(SEQ ID NO: 26) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGAATTGCG-3′; Comp15-48: (SEQ ID NO: 27)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; F50-73 (SEQ ID NO: 28)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGGAAATCA AAA-3′; Comp15-73:(SEQ ID NO: 29) 5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-3′; F50-97:(SEQ ID NO: 30) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGAACAAACTAT-3′; Comp15-97: (SEQ ID NO: 31)5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-3'; F50-120: (SEQ ID NO: 32)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTACAAACA GTT-3′; Comp15-120:(SEQ ID NO: 33) 5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-3′; F50-144:(SEQ ID NO: 34) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTTCCTATTATT-3;; Comp15-144: (SEQ ID NO: 35)5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-3′; F50-169: (SEQ ID NO: 36)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTCACCTG AAA-3′; Comp15-169:(SEQ ID NO: 37) 5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-3′.

In some aspects, this invention provides for a RNA nanostructure robot,wherein the second strand of fastener DNA comprises a sequence which ispartially complementary to the sequence of the first strand.

In some aspects, the moiety R₄ is an aptamer that specifically binds atarget molecule and comprises domain which comprises a sequence which ispartially complementary to the sequence of the second strand, and m isan integer from 1 to 20.

In some aspects, one or more of the R₃ and/or R₄ moieties is an RNAtargeting strand, wherein each targeting strand is operably linked to atargeting moiety and to NR₁ or NR₂. The targeting moiety can be a moietywhich binds to a target. In some aspects, the targeting moiety isselected from: an aptamer that specifically binds a target molecule. Insome aspects, the targeting moiety is an antibody. In some aspects thetarget molecule is a peptide, a protein, an antibody, a glycan, a DNA orRNA sequence, or combinations thereof. In some aspects, targeting moietyis an antibody or fragment thereof, nanobody, receptor or binding domainthereof, aptamer, scFv, fusion protein, or bispecific antibody. In someaspects, the one or more of the R₃ and/or R₄ moieties is an aptamerspecific for nucleolin. In some aspects, the one or more of the R₃and/or R₄ moieties is an aptamer or antibody specific to a targetselected from: interferon (including or excluding interferon-a/b, andinterferon-gamma), a checkpoint inhibitor protein, EGFR, hTNFα, Vacciniavirus, ICAM-1, PDGF-B, VEGF, Nucleolin, Periostin, Vimentin, CEA, AGE,NF-κB, OPN, HGC-27, PSMA, E-selectin, 4-1 BB, OX40, CD28, PSMA/4-1BB,PD-1, PD-L1, IL10R, IL4Ra, CD44/EpCAM, TIM3, CTLA-4, CXCL12, Tenascin-C,Axl, HGC-27, hnRNP Ai, CD16a/c-Met, or VEGF/4-1BB. In some aspects, theaptamer that is specific for nucleolin is an F50 AS1411 aptamer havingthe sequence: 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′ (SEQ ID NO: 38). In someaspects, the targeting strand comprises a domain comprising apolynucleotide sequence for attaching to NR₁ or NR₂. In some aspects,when the nucleolin-specific aptamer is presented to nucleolin on a tumorcell surface, the aptamer will competitively bind to the surface-boundnucleolin. In some aspects, when the RNA nanostructure scaffold is inthe form of a tube comprising a fastener strand wherein the fastenerstrand is a nucleolin-specific aptamer, when the aptamer competitivelybinds to the tumor cell surface-bound nucleolin, the fastener strandwill release from one or all of the RNA nanostructure scaffolds whereinthe scaffold will change shape from a tube to an open rectangular sheet.

In some aspects, this invention provides for a RNA nanostructure robot,wherein the first or second scaffold is configured to have a rectangularsheet having four corners and is shaped into a tube-shape. In someaspects, the dimension of the rectangular sheet can be about 90 nm×about60 nm×about 2 nm. In some aspects, one or more targeting strands arepositioned at one or more corners of the rectangular sheet. In someaspects, the tube-shaped origami structure has a diameter of about 19nm.

In some aspects, one or more of the R₃ and/or R₄ moieties is a capturestrand. In some aspects, the capture strand can bind to a poly(A) regionin NR₁ or NR₂. In some aspects, the capture strands can be operablylinked to a therapeutic agent. In some aspects, the capture strandcomprises an RNA loop. In some aspects, the capture strand comprisespoly(U). In some aspects, the stand comprises a sequence comprising anamino-modified ribonucleoside.

In some aspects, this invention provides for a RNA nanostructure robot,wherein the ssRNA sequence comprises a modified ribonucleic acid. Theribonucleic acid can comprise an alkyl amine functional group. In someaspects, the amino-modified ribonucleoside is incorporated into thessRNA sequence by the addition of 5-Aminoallyluridine-5′-Triphosphateduring a transcription step of forming the ssRNA sequence.

In some aspects, one or more of the R₃ and/or R₄ moieties is an agent.In some aspects, the agent is a therapeutic agent. In some aspects, thetherapeutic agent is a protein. The protein can be selected fromthrombin, prothrombin, or mixtures thereof. In some aspects, thethrombin is conjugated to the amino-modified ribonucleotide by means ofa sulfosuccinimidyl-4-(N-maleimidomethyl) (sulfo-SMCC)cyclohexane-1-carboxylate (sulfo-SMCC) as a bifunctional crosslinker.The maleimide group on the sulfo-SMCC can react to a cysteine on anon-reduced or reduced form of the thrombin molecule, and thesulfosuccinimidyl group on the sulfo-SMCC can react to theamino-modified ribonucleoside.

In some aspects, NR₁ or NR₂ further comprises a complex comprising anRNA nanostructure and at least one therapeutic agent operably linked tothe RNA nanostructure.

In some aspects, the RNA nanostructure comprises one single-stranded RNA(ssRNA) molecule which forms at least one paranemic cohesion crossover.

In some aspects, this invention provides for a RNA nanostructure robot,wherein the RNA nanostructure is immuno-stimulatory.

In some aspects, NR₁ and/or NR₂ comprises a nucleic acid sequence havingat least about 90% sequence identity to SEQ ID NO:1 or SEQ ID NO: 9. Insome aspects, NR₁ and/or NR₂ comprises a nucleic acid sequence having atleast about 95% sequence identity to SEQ ID NO:1 or SEQ ID NO: 9. Insome aspects, NR₁ and/or NR₂ comprises SEQ ID NO:1 or SEQ ID NO: 9. Insome aspects, NR₁ and/or NR₂ consists of SEQ ID NO: 1 or SEQ ID NO: 9.

In some aspects, one or more of R₃ and/or R₄ is a peptide. In someaspects, the peptide comprises a positively-charged moiety. In someaspects, the positively-charged moiety is an amino acid. In someaspects, the positively-charged moiety comprises about 10positively-charged amino acids. The the positively-charged moiety can bea peptide comprising 10 lysine residues.

In some aspects, one or more of the R₃ and/or R₄ moieties is a protein.In some aspects, the protein is selected from: tumor targeting peptide(TTP), a human cancer peptide, or calreticulin protein. In some aspects,the protein is calreticulin protein to RNA-origami to engageinteractions between tumor cells and macrophages or dendritic cells forenhanced antigen presentation and stimulation of antigen-specific Tcells. In some aspects, the protein is Human cancer peptide NY-ESO-1 orMuc1. In some aspects, the TTP is CTKD-K10 having the sequence:

(SEQ ID NO: 3) CTKDNNLLGRFELSGGGSKKKKKKKKKK.

In some aspects, this invention provides for a pharmaceuticalcomposition comprising a RNA nanostructure robot described herein and apharmaceutically acceptable carrier. In some aspects, the pharmaceuticalcomposition further comprises at least one therapeutic agent.

In some aspects, this invention provides for a method of treating cancerin a subject, comprising administering to the subject a therapeuticallyeffective amount of the composition described herein. In some aspects,the cancer is breast cancer, ovarian cancer, melanoma or lung cancer.

In some aspects, this invention provides for a method of inhibitingtumor growth in a subject, comprising administering to the subject atherapeutically effective amount of a composition described herein.

In some aspects, this invention provides for the use of the RNAnanostructure robot as described herein or a composition as describedherein for the manufacture of a medicament for inducing a tumor necrosisresponse in a subject.

In some aspects, this invention provides for the use of the RNAnanostructure robot as described as described herein or a composition asdescribed herein for inducing a tumor necrosis response.

In some aspects, this invention provides for the use of the RNAnanostructure robot as described as described herein or a composition asdescribed herein for the manufacture of a medicament for treating adisease or disorder in a subject.

In some aspects, this invention provides for the use of the RNAnanostructure robot as described as described herein or a composition asdescribed herein for the prophylactic or therapeutic treatment a diseaseor disorder.

As described herein, in certain aspects, the present invention providesan RNA nanostructure robot (also referred to herein as “RNA origami” or“OG-RNA” or “RNA-OG”) which comprises:

-   -   a first nano-robot comprising a single stranded RNA (ssRNA) of        about 1500 to 10,000 bases in length that self-assembles into a        first scaffold; a second nano-robot comprising a ssRNA of about        1500 to 10,000 bases in length that self-assembles into a second        scaffold; and a linker, wherein the linker operably links the        first nano-robot to the second nano-robot.

In certain aspects, the present invention provides a pharmaceuticalcomposition comprising the RNA nanostructure robot as described hereinand a pharmaceutically acceptable carrier.

In certain aspects, the present invention provides a method of treatinga disease or disorder in a subject, comprising administering to thesubject a therapeutically effective amount of the RNA nanostructurerobot or a composition as described herein.

In certain aspects, the present invention provides a method ofinhibiting tumor growth in a subject, comprising administering to thesubject a therapeutically effective amount of the RNA nanostructurerobot or composition as described herein.

In certain aspects, the present invention provides a use of the RNAnanostructure robot or composition as described herein for themanufacture of a medicament for inducing a tumor necrosis response in asubject.

In certain aspects, the present invention provides a use of the RNAnanostructure robot or composition as described herein for inducing atumor necrosis response.

In certain aspects, the present invention provides a use of the RNAnanostructure robot or composition as described herein for themanufacture of a medicament for treating a disease or disorder in asubject.

In certain aspects, the present invention provides a use of the RNAnanostructure robot or composition as described herein for theprophylactic or therapeutic treatment of a disease or disorder.

In certain aspects, the present invention provides a kit comprising theRNA nanostructure robot or composition as described herein andinstructions for administering the RNA nanostructure robot/compositionto a subject to induce an immune response or to treat a disease ordisorder.

The invention also provides processes disclosed herein that are usefulfor preparing an RNA nanostructure described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. RNA origami schematics (left panel) and AFM images (rightpanel). A plasmid containing a ssRNA origami gene was linearized and thessRNA was in vitro transcribed using T7 RNA polymerase. The purified RNAmolecule was self-assembled into the ssRNA rectangle origaminanostructure through paranemic cohesion crossover.

FIG. 2. Nuclease resistance. Self-assembled RNA origami is resistant toRNase I digestion, while the unassembled RNA molecule can be digestedeasily by RNase I. Lane 1 represents 1 kb dsDNA marker. 1 ug ofunassembled RNA molecule was treated without RNase I (lane 2) or with 1U of RNase I for 10 min or 30 min (lane 3 and 4) at room temperature.The self-assembled RNA origami was also treated without RNase I (lane 5)or with 1 U RNase I for 10 min or 30 min (lane 6 and 7) at roomtemperature. The results show that for the unassembled (notnanostructured) RNA molecule, the complex is digested by RNase I. Theself-assembled (nanostructured) RNA origami, however, did notsignificantly change in mobility after RNAse I exposure, indicating thatthe nanostructured RNA origami is more stable than the unassembledsequence.

FIG. 3A shows the ex vivo splenocyte stimulation at 1 day poststimulation. FIG. 3B shows the ex vivo splenocyte stimulation at 2 dayspost stimulation. CD69 activation in T cell. RNA origami activate bothCD8 and CD4 T cells. Percentages of CD69+ cells in CD4 T cells and CD8 Tcells are plotted. PBS: phosphate buffer saline, LPS:lipopolysaccharide; PMB: polymyxin B; Inosine_1: inosine-incorporatedRNA origami. For each grouping, CD4 T cell is shown in the left and CD8T cell is shown on the right.

FIG. 4A shows the ex vivo splenocyte stimulation. FIG. 4B shows ex vivosplenocte stimulation in plasmacytoid DC. CD86 activation in Dendriticcell. RNA origami activate antigen presenting cells (DC and plasmacytoidDC (pDC)). Mean fluorescence intensity of CD86 in each cell populationis plotted.

FIG. 5A shows cytokine release in ex vivo splenocyte cell culturesupernatant upon stimulation, where RNA origami induces production ofIFN-α. FIG. 5B shows cytokine release in ex vivo splenocyte cell culturesupernatant upon stimulation, where RNA origami induces production ofIFN-b.

FIG. 6. Serum cytokine in mice injected with RNA origami. Similar to thefinding on in vitro stimulation, an intravenous injection of RNA origamithrough retro-orbital route resulted in a transient elevation of IFNa/b.

FIG. 7. Influence on tumor cell viabilities. After three days ofincubation, RNA origami was found to reduce the viability of 4T1, amouse breast cancer cell line, in vitro. The delayed inhibitory effectmight have been mediated through the production of pro-inflammatorycytokines by the tumor cells after their exposure to RNA origami. RNAorigami exerted little or minimal effect on the viability of certainother mouse and human tumor cell lines tested in vitro (not shown).Within each grouping, the following are included from left to right: NT(no treatment), RNA (RNA origami) 5 μg/ml, RNA (RNA origami) 0.5 μg/ml,RNA (RNA origami) 0.05 μg/ml, polyIC 5 μg/ml, polyIC 0.5 μg/ml, polyIC0.05 μg/ml, +(camptothecin).

FIG. 8. TLR3 agonist. RNA origami showed as a TLR3 agonist in a reportercell line, HEK-Blue™-mTLR3 cells, although its activity is not as strongas polyIC.

FIG. 9A shows the anti-tumor immunity in vivo where the antibody only isshown.

FIG. 9B shows the anti-tumor immunity in vivo where the RNA origami+antibody is shown. Track in vivo tumor growth with A20-iRFP model.

FIG. 10. Tumor reduction upon treatment with RNA origami.

FIG. 11. MuLE (Multiple Leniviral Expression) Destination Vector.

FIG. 12. Cytokines and chemokines three hours after IP treatment. PBS isthe left columns in each cytokine set; RNA-OG is the middle column ineach cytokine set, and poly IC is the right column in each cytokine set.

FIG. 13A shows the treatment and scheduling of mice with certainembodiments of the RNA nanostructures (“RNA-origami”) described herein.FIG. 13B shows lack of inhibition of tumor growth in control. FIG. 13Cshows inhibition of tumor growth in control after RNA-origami injection.Mice treated with RNA-origami show significant reduction in tumorgrowth.

FIG. 14. RNA-origami increases pro-inflammatory cytokines and reducesanti-inflammatory cytokines. The high levels of IFNg and TNFa in thebearing-tumor bearing mouse treated with RNA-origami clearly showedstrong induction of adaptive anti-tumor immunity. The left column (whichis not visible in the Figure) is the biomarker levels in normal serum.The middle column is the biomarker levels of ascites fluid from tumorbearing mice. The right-most column is the biomarker levels of ascitesfluid from RNA-origami treated tumor bearing mice. The resultsdemonstrate that the RNA origami results in increases of anti-tumor(pro-inflammatory) cytokines.

FIG. 15. Schematic of functions of HSP70 protein and derived peptides(also referred to as “TPP” or “TKD”).

FIG. 16. Different RNA-OG/TTP ratios lead to different sizes ofcomplexes. The complex appear stable after its formation as the old andnew complexes formed at 1:200 (molar) ratios displayed similar patternof mobility (lane 3 and lane 7).

FIG. 17. Different complexes exhibit different binding/internalizationprofiles, as shown by flow cytometry. Higher internalization ofRNA-origami (OG) by RAW cells than CT-26. Upon increase amount of thepeptide, the lower level of binding to both CT-26 and RAW cells.

FIG. 18. Red-fluorescence positive tumor cells were inoculated at day 0and tumor nodule formed on day 9 (i.e., pretreatment). These mice werethen treated with a single injection of different types of RNAstructures, free RNA or RNA-origami coated with tumor-targeting peptide(TTP). The mice were monitored for more than 20 days, and tumorregression was found in the mouse receiving the RNA-Origami polymer, butnot other groups (including RNA-origami only group).

FIG. 19A-FIG. 19C: FIG. 19A shows memory recalled responses. Splenocytesstimulated in vitro by PBS (Buffer), TPP, RNA-OG-TPP, Irrelevant (KLH)peptide, or RNA-OG;

FIG. 19B shows representative ELISPOT readout, where each spotrepresents an IFNg-producing immune cell that was activated by differentstimuli; and FIG. 19C shows quantification of ELISPOTS. Tumor-free mousedeveloped TPP-specific immunity as revealed by ELISPOT assay, in whichTTP-stimulated splenocytes produced IFNg after the splenocyte culturedwith TPP, but not irrelevant peptides.

FIG. 20. Exemplary RNA nanostructure double-robot. In some embodiments,RNA-origami comprises antigen-specific peptides, which are linked via apH-sensitive double helix to an RNA-nanocage that is responsive tointerferon-gamma to release checkpoint inhibitor antagonists.

FIG. 21 depicts an unfastened rectangular RNA origami structure havingfasteners extending from the edges that can be joined,aptamer-containing targeting strands and antigen-specific peptides (toinduce tumor-specific immunity), as well as pH-sensitive linker to allowdissociation of RNA-origami from the RNA nanocages in tumormicroenvironment.

FIG. 22 depicts an aptamer-containing targeting strand containing anaptamer portion and an attaching RNA strand.

FIG. 23 depicts an aptamer-containing targeting strand containing anaptamer portion and an attaching RNA strand, and having a quenchermoiety attached to one arm of the Y-structure and a fluorophore moietyattached to the second arm of the Y-structure.

FIG. 24 depicts a therapeutic agent-RNA conjugate capture strand havinga ssRNA attachment strand and a therapeutic agent payload.

FIG. 25 depicts a therapeutic agent-RNA conjugate capture strand havinga ssRNA attachment strand and a therapeutic agentpayload, where thetherapeutic agentpayload is operably linked to an imaging agent.

FIG. 26 depicts a therapeutic agent-RNA conjugate capture strand havinga ssRNA attachment strand that is a pH-sensitive linker.

FIG. 27 depicts a drug-RNA conjugate capture strand having a ssRNAattachment strand that is linked to a therapeutic agentpayload by meansof a linker, where the therapeutic agentpayload is operably linked to animaging agent.

FIG. 28 depicts an unfastened rectangular RNA origami structure havingfour therapeutic agent-RNA conjugates operably linked to the origamistructure. The therapeutic agent-RNA conjugates can be attached toeither the “top” or the “bottom” (or both) of the origami structure,such that when the origami structure is rolled into a tube, the drug-RNAconjugates can be designed to be either on the inside or outside of thetube.

FIG. 29 depicts an unfastened rectangular RNA origami structure havingthree drug-RNA conjugates operably linked to the origami structure. Thedrug-RNA conjugates can be attached to either the “top” or the “bottom”(or both) of the origami structure, such that when the origami structureis rolled into a tube, the drug-RNA conjugates can be designed to beeither on the inside or outside of the tube.

FIG. 30 depicts an unfastened rectangular RNA origami structure havingtwo drug-RNA conjugates operably linked to the origami structure. Thedrug-RNA conjugates can be attached to either the “top” or the “bottom”(or both) of the origami structure, such that when the origami structureis rolled into a tube, the drug-RNA conjugates can be designed to beeither on the inside or outside of the tube.

FIG. 31 depicts an unfastened rectangular RNA origami structure havingone drug-RNA conjugate operably linked to the origami structure. Thedrug-RNA conjugate can be attached to either the “top” or the “bottom”of the origami structure, such that when the origami structure is rolledinto a tube, the drug-RNA conjugate can be designed to be either on theinside or outside of the tube.

FIG. 32 depicts an exploded view of the RNA origami structure, detailingthe hybridization of a single stranded RNA scaffold strand and staplestrands, and the interaction of the two staple strands.

FIG. 33 depicts a tube-shaped RNA origami structure having drug-RNAconjugates positioned on the outside of the tube-shaped RNA origamistructure.

FIG. 34 depicts a tube-shaped RNA origami structure having drug-RNAconjugates positioned on the inside of the tube-shaped RNA origamistructure.

FIG. 35 depicts a tube-shaped RNA origami structure having drug-RNAconjugates positioned on the inside of the tube-shaped RNA origamistructure, having aptamer-containing targeting strands positioned at theends of the tube, and illustrating the fasteners joining the edges ofthe RNA origami structure so as to form a tube shape.

FIG. 36 depicts a schematic for an RNA nanostructure double robot whereNR₁ and NR₂ are separate ssRNA scaffolds linked through a linker eitherdirectly via direct partial hybridization or indirectly through abridging oligonucleotide.

FIG. 37 depicts nondenaturing gel electrophoresis results of ssRNAnanostructure-peptide complexes demonstrating the solubility andstability of the complexes.

FIG. 38 depicts a molecular model of some embodiments of the ssRNAscaffold (“RNA cage”) wherein the scaffold can fold into a tubularshape. The RNA cage can comprise IFN-γ aptamers, pH-sensitive linkers,anti-PD1 aptamers, anti-PD1 antibodies, and/or nucleolin-bindingaptamers. The RNA cage can close in the presence of IFN-γ compoundswhereby the IFN-γ aptamers located at the periphery of the RNA cagecommonly bind to a single IFN-γ compound. In the presence of excess freeIFN-γ compounds, the IFN-γ aptamers will each bind to the IFN-γcompounds, opening the cage to expose any moieties internal to the cage.

DETAILED DESCRIPTION

Double-stranded RNA (dsRNA) is a by-product of viral infection. It is anatural ligand of Toll-like receptor 3 (TLR3) and a potent stimulatorfor activating innate and adaptive immunity. PolyIC is a synthetic dsRNAanalogue and has been widely explored for anti-cancer immunotherapy.PolyIC, however, is associated with high toxicity, primarily due toexcessive production of cytokines, which subsequently leads tocytokinemia. The adjuvant activity of RNA-origami was tested in cancerimmunotherapy and it was found that repeat injections (e.g., 2, 3, 4, 5,6, 7, or 8 injections, depending on the tumor load and intrinsic tumorimmunogenicity) of RNA-origami at 16 μg/dose significantly delayed tumorgrowth. In certain embodiments, the dosage is less than about 5 mg/kg,less than about 4 mg/kg, less than about 3 mg/kg, less than about 2mg/kg, less than about 1 mg/kg, or less than about 0.8 mg/kg. Inaddition, when the cytokine profiles were analyzed, it was found thatthe cytokines produced by the mice treated with RNA-origami had higherlevels of particular cytokines and chemokines required for thegeneration of effective anti-tumor immunity, but lower levels ofcytokines involved in systemic cytokine storm. Thus, RNA-origami can beused as effective and safe adjuvants. Further, it was demonstrated thatthe present RNA-origami exhibit potent anti-tumor activity, but withoutapparent toxicity.

TLR3 ligands have multiple modes of action in cancer therapy. They canbe used as inducers of apoptosis/neprotosis in cancer cells. They arestrong activators for the production of type-I interferon in a widerange of cell types, including host immune cells and cancer cells, viatwo major pathways: TLR3 (endo-lysosome) and MDA/RIG (present incytoplasm). TLR3 ligands exhibit synergistic effects in combination withchemotherapeutics, apoptosis enhancers, other TLR ligands, tumorantigens, and checkpoint inhibitors (e.g., anti-PD1, CTLA4 or PD-L1).The same ligands are used for both murine models and for humans.

TLR3 ligands include PolyIC, Poly A:U and ARNAX. There are three typesof PolyIC: (1) standard Poly-IC, which is rapidly inactivated by serum;(2) Poly-IC/poly-lysine (polyICLC, Hiltonol, Oncovir), which has beenstudied in 12 clinical trials for many malignant tumors; and (3)Poly(I:C12U) (Ampiligen), which has been studied in clinical trials forOVC and peritoneal tumors. PolyIC has been tested in humans since thelate 1970s as anti-cancer adjuvants. PolyIC, however, was found to bequickly inactivated by serum. Although its complex with poly-lysinegreatly enhances its half-life in circulation and efficacy, complexedpolyIC causes intolerable adversity, due to excessive production ofcytokines. It is believed that polyIC activates both TLR3 and MDA5/RIGsignaling pathways. The latter has been linked to systemic toxicity.Instead, polyIC has been explored as a part of cancer vaccines by mixingwith tumor-specific antigens, which were delivered locally.

In addition, double-stranded poly A:U was tested in early 1980s inclinical studies. Due to its low efficacy (possibly labile) and poorcellular uptake, the efforts were discontinued. Recently, the intereston poly A:U investigation has been renewed because it was found thatpoly A:U only activates TLR3, but not the MDA/RIG signaling pathways,therefore causing low toxicity.

A third line of study involves ARNAX, which is phosphorothioateODN-guided dsRNA (sODN-dsRNA) that resembles PolyA:U. It exhibitsODN-mediated cellular uptake. (Matsumoto, M. et al. 2015. NatureCommunications. 6:6280) RNA-origami is advantageous over other TLR3ligands for many reasons. For example, they are scalable in terms ofquantity for production with relatively low cost. They have well-definedstructure and uniformity for reproducibility. The particulate size andintrinsic RNA nanoparticle robot structure as described herein issuperior for better internalization by immune cells without additionalpackaging to promote phagocytosis, like the processes involved inpolyIC, dsRNA or the synthetic oligo-DNA-RNA hybrid (i.e., ARNAX). ARNAXactivates endosomal toll-like receptor 3 (TLR3), but not cytoplasmicMDA5/RIG-I. The TLR3-TICAM-1-IRF3-IFN-β signaling axis is indispensablein dendritic cells (DCs) for ARNAX-mediated cytotoxic T lymphocyte (CTL)induction. (B) ARNAX therapy enhances antitumor responses in conjunctionwith PD-1/PD-L1 blockade. Tumors are self-originating and essentiallylack an adjuvant. In the absence of adjuvant, DCs remain immature state(immature DC) and fail to induce tumor-associated antigen (TAA)-specificCTLs. ARNAX activates TLR3 in DCs to induce maturation and cross-primingof TAA-specific CTLs in lymphoid tissues. They are highly stable so asto be feasible for in vivo applications. The RNA nanoparticle robots asdescribed herein have better safety as they may selectively activate apathway (TLR3) that is required for an induction of adaptive cellularimmunity (anti-cancer or anti-viral), but not MDA5/RIG pathway, andtherefore are less likely to induce a cytokine storm. The RNAnanoparticle robots as described herein have well-defined structure anduniformity for reproducibility, unlike heterogenous population of polyIC(low vs high molecular weight) with different functional activities.Thus, the RNA nanoparticle robots as described herein have surprisingstability, uptake, homogeneity, selectivity and low toxicity.

RNA Nanostructure Complexes

In some embodiments, this disclosure includes a RNA nanostructure robot(also referred to herein as “RNA origami” or “OG-RNA” or “RNA-OG”)having the sequence of (R₃)_(n)-NR₁-L-NR₂-(R₄)_(m), wherein:

-   -   NR₁ represents a first nano-robot comprising a single stranded        RNA (ssRNA) of about 1500 to 10,000 bases in length that        self-assembles into a first scaffold;    -   NR₂ represents a second nano-robot comprising a ssRNA of about        1500 to 10,000 bases in length that self-assembles into a second        scaffold;    -   L is a linker which operably links NR₁ to NR₂;    -   wherein R₃ and R₄ are independently selected from a pair of        fastener strands, an aptamer, a cargo molecule, a capture        strand, a targeting strand, or H;    -   n is an integer from 0 to 50; and    -   m is an integer from 0 to 50.

In certain embodiments, n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In certain embodiments,m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, and 20. When n or m is 0, the R₃ or R₄ moiety is notpresent. When n is 2 or greater, there can be multiple separate R₃moieties simultaneously present (e.g., one R₃ is an aptamer, another R₃is a cargo molecular, another R₃ is a capture strand, etc.). When m is 2or greater, there can be multiple separate R₄ moieties simultaneouslypresent (e.g., one R₄ is an aptamer, another R₄ is a cargo molecular,another R₄ is a capture strand, etc.).

In certain embodiments, the linker is an oligonucleotide. Theoligonucleotide can be comprised of DNA, RNA, modified DNA, modifiedRNA, or combinations thereof.

In certain embodiments, the present invention provides a complexcomprising an RNA nanostructure and at least one diagnostic and/ortherapeutic agent operably linked to the RNA nanostructure.

In certain embodiments, the RNA nanostructure comprises onesingle-stranded RNA (ssRNA) molecule, wherein the ssRNA molecule formsat least one paranemic cohesion crossover, and wherein the RNAnanostructure has immuno-stimulatory properties.

In certain embodiments, the RNA nanostructure comprises onesingle-stranded RNA (ssRNA) molecule, wherein the ssRNA moleculecomprises a plurality of regions of double helices, wherein at least twoof the plurality of regions of double helices form a paranemic cohesioncrossover, and wherein the RNA nanostructure has immuno-stimulatoryproperties.

As used herein, the term “RNA nanostructure” refers to a nanoscalestructure made of RNA, wherein the RNA acts both as a structural andfunction element. RNA nanostructures can also serve as a scaffold forthe formation of other structures. RNA nanostructures may be prepared bymethods using one or more nucleic acid oligonucleotides. For example,such nanostructures may be assembled based on the concept ofbase-pairing. While no specific sequence is required, the sequences ofeach oligonucleotide must be partially complementary to certain otheroligonucleotides to enable hybridization of all strands or sequenceswithin a single oligonucleotide to enable hybridization and assembly ofthe nanostructure. For example, in certain embodiments, the RNAnanostructure is an RNA rectangle origami nanostructure, self-assembledfrom one single-stranded RNA molecule through paranemic cohesioncrossover.

As used herein, the term “nanorobot” or “RNA nanostructure robot” or“DNA nanostructure cage” refers to a RNA or DNA nanostructure whichexhibits at least one function, which can be active and/or passive. Thefunction can include or exclude delivery of a moiety R₃ or R₄. In someembodiments one or more of the R₃ and/or R₄ moieties is an agent. ActiveRNA nanostructure functions can include cage opening, wherein a foldedRNA nanostructure opens in the presence of a stimulus. In someembodiments, the stimulus is the presence of a high localizedconcentration of a competitive binding partner to an aptamer or antibodypresent at the periphery of the RNA nanostructure sheet. Passive DNAand/or RNA nanostructure functions can include carrying and/or deliveryof agents, either covalently or non-covalently. Passive nanostructurefunctions may also include release of a carried agent upon a stimulus.Covalently carrying agents can occur through the cross-linking of anamino-modified ribonucleotide with a peptide or protein through abifunctional compound. The bifunctional compound can be sulfo-SMCC.

Non-covalently carrying agents can occur by hybridization of an agentcomprising an oligonucleotide sequence which is partially complementaryto the RNA nanostructure sequence. In some embodiments, delivery ofagents can comprise encapsulation of an agent within a DNA cage.

In certain embodiments, the RNA nanostructure can be formed without theuse of “staple strands.” It was surprisingly found that in someembodiments, the RNA nanostructure can self-assemble with no addedstable strands. In some embodiments, the DNA nanocage comprises staplestrands. As used herein, “staple strands” are short single-strandedoligonucleotides of about 20 to about 40 nucleotides in length, such as20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, or 40 nucleotides in length, wherein one end of the staplestrand hybridizes with a region of the scaffold strand, and the secondend of the staple strand hybridizes with another region of the scaffoldstrand, thereby “stapling” the two regions of the scaffold strand. Incertain embodiments, the dimension of an RNA nanostructure sheet formedwithout the use of staple strands is about 90 nm×about 60 nm×2 nm.

In certain embodiments, one of more of R₃ and/or R₄ is an agents whichmay be operably linked to the RNA nanostructure, such as diagnosticagents or therapeutic agents. In certain embodiments, at least onediagnostic agent is operably linked to the RNA nanostructure. In certainembodiments, at least one therapeutic agent is operably linked to theRNA nanostructure. In certain embodiments, at least one diagnostic agentand at least one therapeutic agent are operably linked to the RNAnanostructure.

As used herein, the term “paranemic cohesion crossover” refers to afour-stranded nucleic acid complex containing a central dyad axis thatrelates two flanking parallel double helices” (Zhang et al. J. Am Chem.Soc. 2002). The strands are held together exclusively by Watson-Crickbase pairing. The key feature of the structure is that the two adjacentparallel nucleic acid double helices form crossovers at every pointpossible. Hence, reciprocal crossover points flank the central dyad axisat every major or minor groove separation. In other words, paranemiccohesion crossover refers to Watson-Crick base pairing interactionsbetween two parallel double helices comprising a central dyad axis.

The assembly of such RNA nanostructures may be based on base-pairingprinciples or other non-canonical binding interactions. For example,while no specific RNA sequence is required, regions of complementarywithin a single RNA molecule or between multiple RNA molecules may beused for assembly. Persons of ordinary skill in the art will readilyunderstand and appreciate that the optimal sequence for any given RNAnanostructure will depend on the desired or intended shape, size,nucleic acid content, and intended use of such RNA structure. In certainembodiments, wherein the nanostructure comprises more than one ssRNAmolecule (e.g. two or more oligonucleotides/polynucleotides), each ssRNAmolecule may have a region that is complementary to a region on anotherssRNA molecule to enable hybridization of the strands and assembly ofthe nanostructure. In certain other embodiments, wherein thenanostructure consists of a single ssRNA molecule (i.e., a singleunimolecular RNA oligonucleotide/polynucleotide), regions within themolecule may be complementary to certain other regions within themolecule to enable hybridization and assembly of the nanostructure. RNAnanostructures produced in accordance with the present disclosure aretypically nanometer-scale structures (e.g., having length scale of 1 to1000 nanometers), although, in some instances, the term “nanostructure”herein may refer to micrometer-scale structures (e.g., assembled frommore than one nanometer-scale or micrometer-scale structure). In someembodiments, a RNA nanostructure described herein has a length scale of1 to 1000 nm, 1 to 900 nm, 1 to 800 nm, 1 to 700 nm, 1 to 600 nm, 1 to500 nm, 1 to 400 nm, 1 to 300 nm, 1 to 200 nm, 1 to 100 nm or 1 to 50nm. In some embodiments, a RNA nanostructure described herein has alength scale of greater than 1000 nm. In some embodiments, a RNAnanostructure described herein has a length scale of 1 micrometer to 2micrometers.

In certain embodiments, the RNA nanostructure comprises, consistsessentially of, or consists of multiple ssRNA molecules (e.g., more thanone oligonucleotide/polynucleotide strands, such as two or more ssRNAmolecules). In certain embodiments, the RNA nanostructure comprises twoor more ssRNA molecules, which are capable of self-assembling (orconfigured to self-assemble) into a nanostructure. In certainembodiments, the RNA nanostructure is assembled from two or more ssRNAmolecules through paranemic cohesion crossovers. Thus, in certainembodiments, the RNA nanostructure comprises two or more ssRNAmolecules, wherein the ssRNA molecules self-assemble to form at leastone paranemic cohesion crossover.

In certain embodiments, the RNA nanostructure comprises, consistsessentially of, or consists of a single ssRNA molecule (i.e., oneunimolecular oligonucleotide/polynucleotide strand). In certainembodiments, the RNA nanostructure is assembled using one ssRNA molecule(e.g., in certain embodiments one and only one, exactly one, or greaterthan zero and less than two).

In certain embodiments, the RNA nanostructure is comprised of one ssRNAmolecule, which is capable of self-assembling into a nanostructure. Incertain embodiments, the RNA nanostructure consists of one ssRNAmolecule, which is capable of self-assembling into a nanostructure. Incertain embodiments, the RNA nanostructure is assembled from one ssRNAmolecule through paranemic cohesion crossovers. Thus, in certainembodiments, the RNA nanostructure comprises one single-stranded RNA(ssRNA) molecule, wherein the ssRNA molecule forms at least oneparanemic cohesion crossover.

The length of each RNA strand is variable and depends on, for example,the type of nanostructure to be formed. In certain embodiments, the RNAnanostructure is comprised of multiple oligonucleotide strands. Incertain embodiments, the RNA nanostructure is comprised of a single(i.e., unimolecular) oligonucleotide strand. In certain embodiments, theoligonucleotide or RNA strand is about 15 nucleotides in length to about150,000 nucleotides in length, about 15 to about 7500 nucleotides inlength, about 3000 to about 7000 nucleotides in length, about 5000 toabout 7000 nucleotides in length, about 5500 to about 6500 nucleotidesin length, about 15 to about 5000 nucleotides in length, about 15 toabout 4000 nucleotides in length, about 15 to about 3000 nucleotides inlength, about 250 to about 3000 nucleotides in length, about 500 toabout 3000 nucleotides in length, about 1000 to about 3000 nucleotidesin length, about 1500 to about 2500 nucleotides in length, or betweenany of the aforementioned nucleotide lengths. In certain embodiments,the at least one ssRNA molecule (i.e., oligonucleotide or RNA strand) isabout 10 nucleotides in length to about 200,000 nucleotides in length,the at least one ssRNA molecule (i.e., oligonucleotide or RNA strand) isabout 10 nucleotides in length to about 100,000 nucleotides in length,the at least one ssRNA molecule (i.e., oligonucleotide or RNA strand) isabout 10 nucleotides in length to about 90,000 nucleotides in length,about 10 to about 80,000 nucleotides in length, about 10 to about 70,000nucleotides in length, about 10 to about 60,000 nucleotides in length,about 10 to about 50,000 nucleotides in length, about 10 to about 40,000nucleotides in length, about 10 to about 30,000 nucleotides in length,about 10 to about 25,000 nucleotides in length, or about 10 to about20,000 nucleotides in length. In certain embodiments, the ssRNA molecule(i.e., oligonucleotide or RNA strand) is about 15 nucleotides in lengthto about 20,000 nucleotides in length, the ssRNA molecule (i.e.,oligonucleotide or RNA strand) is about 15 nucleotides in length toabout 10,000 nucleotides in length, about 15 to about 7500 nucleotidesin length, about 3000 to about 7000 nucleotides in length, about 5000 toabout 7000 nucleotides in length, about 1500 to about 6500 nucleotidesin length, about 1000 to about 7000 nucleotides in length, about 5500 toabout 6500 nucleotides in length, about 15 to about 5000 nucleotides inlength, about 15 to about 4000 nucleotides in length, about 15 to about3000 nucleotides in length, about 250 to about 3000 nucleotides inlength, about 500 to about 3000 nucleotides in length, about 1000 toabout 3000 nucleotides in length, or about 1500 to about 2500nucleotides in length. In certain embodiments, the ssRNA molecule (i.e.,oligonucleotide or RNA strand) is about 100, about 200, about 300, about400, about 500, about 600, about 700, about 800, about 900, about 1000,about 1100, about 1200, about 1300, about 1400, about 1500, about 1600,about 1700, about 1800, about 1900, about 2000, about 2100, about 2200,about 2300, about 2400, about 2500, about 2600, about 2700, about 2800,about 2900, about 3000, about 3100, about 3200, about 3300, about 3400,about 3500, about 3600, about 3700, about 3800, about 3900, about 4000,about 4100, about 4200, about 4300, about 4400, about 4500, about 4600,about 4700, about 4800, about 4900, about 5000, about 5100, about 55200, about 5300, about 5400, about 5500, about 5600, about 5700, about5800, about 5900, about 6000, about 6100, about 6200, about 6300, about6400, about 6500, about 6600, about 6700, about 6800, about 6900, about7000, about 7100, about 7200, about 7300, about 7400, about 7500, about7600, about 7700, about 7800, about 7900, about 8000, about 8100, about8200, about 8300, about 8400, about 8500, about 8600, about 8700, about8800, about 8900, about 9000, about 9100, about 9200, about 9300, about9400, about 9500, about 9600, about 9700, about 9800, about 9900, about10000, about 10100, about 10200, about 10300, about 10400, about 10500,about 10600, about 10700, about 10800, about 10900, about 11000, about11000, about 11100, about 11200, about 11300, about 11400, about 11500,about 11600, about 11700, about 11800, about 11900, about 12000, about12100, about 12200, about 12300, about 12400, about 12500, about 12600,about 12700, about 12800, about 12900 nucleotides in length, about 13000nucleotides in length, about 14000 nucleotides in length, about 15000nucleotides in length, about 16000 nucleotides in length, about 17000nucleotides in length, about 18000 nucleotides in length, about 19000nucleotides in length, about 20000 nucleotides in length, about 25000nucleotides in length, about 30000 nucleotides in length, about 35000nucleotides in length, about 40000 nucleotides in length, about 45000nucleotides in length, about 50000 nucleotides in length, about 75000nucleotides in length, about 100000 nucleotides in length, about 125000nucleotides in length, about 150000 nucleotides in length, about 175000nucleotides in length or about 200000 nucleotides in length.

In some embodiments, the RNA is synthesized de novo using chemical orbiological methods. The RNA can be chemically synthesized in a step-wisemanner. In some embodiments, the RNA can be synthesized using thecyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M.H., Tet. Let. 22:1859, 1981); nucleoside H-phosphonate method (Garegg etal., Tet. Let. 27:4051-4054, 1986; Froehler et al., Nucl. Acid. Res.14:5399-5407, 1986; Garegg et al., Tet. Let. 27:4055-4058, 1986, Gaffneyet al., Tet. Let. 29:2619-2622, 1988). These chemistries can beperformed by a variety of automated oligonucleotide synthesizersavailable in the market, including the use of an in vitro transcriptionmethod. In some embodiments, the RNA is synthesized de novo bytranscription.

In some embodiments, the ssRNA can comprise the sequence of:

-   -   (HD₁-LD₁-HD₂-LD₂)_(x)    -   wherein x is selected from 2 to 100, 2 to 500, 2 to 1000, 2 to        1500, 2 to 2000, 2 to 2500, 2 to 3000, or 2 to 3500, 2 to 4000,        2 to 4500, 2 to 5000, 2 to 5500, or 2 to 6000;    -   wherein HD₁ and HD₂ are each an RNA helical domain;    -   wherein LD₁ and LD₂ are each an RNA locking domain;    -   and further wherein the ssRNA sequence, when folded, exhibits at        least one paranemic cohesion crossover. As discussed herein, the        term “helical domain” is used interchangeably with the term “a        region of a double helix”. Additionally, the term “locking        domain” is used interchangeably with the term “paranemic        cohesion crossover”. In certain embodiments, HD₁ and HD₂        independently comprise from about 5 to about 50 ribonucleotides.        In certain embodiments, LD₁ and LD₂ independently comprise from        about 5 to about 50 ribonucleotides.

In certain embodiments, the RNA nanostructure is assembled using asingle stranded RNA molecule. In certain embodiments, the RNAnanostructure comprises both single stranded and double strandedregions. In certain embodiments, the ssRNA molecule comprises at leasttwo parallel double helices. In certain embodiments, the ssRNA moleculescomprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 parallel double helices. In certain embodiments, in theself-assembled RNA nanostructure design, a majority (˜95%) isdouble-stranded region, and only a small portion (˜5%) representssingle-stranded RNA.

In certain embodiments, the RNA nanostructure is comprised of one ssRNAmolecule, which is capable of self-assembling into a nanostructure. Incertain embodiments, the RNA nanostructure is assembled from one ssRNAmolecule through paranemic cohesion crossovers. Thus, in certainembodiments, the RNA nanostructure comprises one single-stranded RNA(ssRNA) molecule, wherein the ssRNA molecule forms at least oneparanemic cohesion crossover. In certain embodiments, the RNAnanostructures has at least one paranemic cohesion crossover. In certainembodiments, the RNA nanostructures has at least one to about 200paranemic cohesion crossovers. In certain embodiments, the RNAnanostructures has at least 12 to about 100 paranemic cohesioncrossovers. In certain embodiments, the RNA nanostructures has at least20 to about 80 paranemic cohesion crossovers. In certain embodiments,the RNA nanostructures has at least 40 to about 60 paranemic cohesioncrossovers. An RNA (e.g., a ssRNA molecule) may be designed to assembleinto a double-stranded chain, resembling a large hairpin structure. Thathairpin structure then assembles to form a structure containing paireddouble helices (or regions thereof) and paranemic cohesion crossovers.In certain embodiments, the RNA nanostructure comprises between about 1to about 200 paranemic cohesion crossovers.

A “layer” of an RNA nanostructure, as used herein, refers to a planararrangement of a portion of the RNA chain. In certain embodiments, anRNA nanostructure comprises 2 or more layers. In some embodiments, anRNA nanostructure may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or morelayers, depending on the desired shape of the nanostructure. In certainembodiments, the paranemic cohesion crossovers direct the RNA chain tofurther assemble into the final structure. The paranemic cohesioncrossovers within the bottom layer are designed to (or configured orsequence-coded to) base pair with their corresponding paranemic cohesioncrossovers within the top layer, but without traversing through eachother. In some embodiments, a nanostructure comprises a first layercomprising a plurality of double helices and a plurality of paranemiccohesion crossovers, wherein at least two regions of double helices ofthe first layer are separated from each other by a paranemic cohesioncrossover, and a second layer comprising a plurality of double helicesand a plurality of paranemic cohesion crossovers, wherein at least tworegions of double helices of the second layer are separated from eachother by a paranemic cohesion crossover, wherein a paranemic cohesioncrossover of the first layer is hybridized to a paranemic cohesioncrossover of the second layer.

In certain embodiments, the shape of the RNA nanostructure can includeor exclude a polyhedron, a tube, a spheroid, or an elliptoid. In certainembodiments, the polyhedron can include or exclude a rectangle, diamond,tetrahedron, or triangle. In certain embodiments, the shape of the RNAnanostructure is, for example, a rectangle, a diamond, a tetrahedron, atriangle, or any other user-defined geometric shape. Persons of ordinaryskill in the art will, after having studied the teachings herein,appreciate and understand that these teachings are not limited to anyspecific RNA nanostructure shape, but rather can be applied to generateany desired shape by programming (or generating) the RNA molecule withthe requisite sequence that will cause the molecule to self-assemblethrough pairing interactions into the desired shape. In certainembodiments, the shape of the RNA nanostructure is a rectangle. Incertain embodiments, the RNA nanostructure is an RNA rectanglenanostructure, self-assembled from one single-stranded RNA moleculethrough paranemic cohesion crossover. In some embodiments, the rectangleRNA nanostructure comprises at least one loop region (e.g., 13 loopsregions). In certain embodiments, the loop regions comprise or consistof a sequence selected from the group consisting of UUUC (SEQ ID NO: 4),GGGAGGG (SEQ ID NO: 5), CCCUCCC (SEQ ID NO: 6), AAAGAAA (SEQ ID NO: 7),and UUUCUUU (SEQ ID NO: 8). In certain embodiments, at least 25% of theloop regions may comprise or consist of UUUC (SEQ ID NO: 4), of GGGAGGG,(SEQ ID NO: 5), of CCCUCCC (SEQ ID NO: 6), of AAAGAAA (SEQ ID NO: 7) orof UUUCUUU (SEQ ID NO: 8). In certain embodiments, at least 50% of theloop regions may comprise or consist of UUUC (SEQ ID NO: 4), of GGGAGGG(SEQ ID NO: 5), of CCCUCCC (SEQ ID NO: 6), of AAAGAAA (SEQ ID NO: 7) orof UUUCUUU (SEQ ID NO: 8). In certain embodiments, at least 75% of theloop regions may comprise or consist of UUUC (SEQ ID NO: 4), of GGGAGGG(SEQ ID NO: 5), of CCCUCCC (SEQ ID NO: 6), of AAAGAAA (SEQ ID NO: 7) orof UUUCUUU (SEQ ID NO: 8). In certain embodiments, all of the loopregions may comprise or consist of UUUC (SEQ ID NO: 4), of GGGAGGG (SEQID NO: 5), of CCCUCCC (SEQ ID NO: 6), of AAAGAAA (SEQ ID NO: 7) or ofUUUCUUU (SEQ ID NO: 8).

In certain embodiments, the RNA nanostructure is a rectangle origaminanostructure. For example, in certain embodiments, the single strandedRNA molecule is SEQ ID NO:1, as described in Example 1. As described inthe Examples, this nanostructure may be surprisingly used as animmune-adjuvant to boost an immune response, including inducinganti-tumor immunity. Advantageously, this adjuvant may be easily scaledup by biochemical production.

Accordingly, certain embodiments of the invention provide an RNAnanostructure comprising a nucleic acid sequence having at least about60% sequence identity to SEQ ID NO:1. In certain embodiments, the RNAnanostructure comprises a nucleic acid sequence having at least about61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO:1. In certain embodiments, the RNA nanostructureconsists of a nucleic acid sequence having at least about 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO:1. In certain embodiments, the RNA nanostructure comprises SEQ IDNO:1. In certain embodiments, the RNA nanostructure consists of SEQ IDNO:1.

In certain embodiments, the RNA nanostructure comprises one or moremodified nucleic acids. The modified nucleic acids can be a modifiedribonucleotide or ribonucleoside. In some embodiments, the modifiedribonucleoside can be selected from the RNA-incorporated from, in part,5-Aminoallyluridine-5′-Triphosphate (Trilink, N-1062), orBiotin-16-Aminoallyluridine-5′-Triphosphate (Trilink, N-5005).

In some embodiments, one or more of R₃ and/or R₄ can be a tumor-specificantigen. In certain embodiments, the tumor-specific antigen is TKD. Itis understood that the tumor-specific antigens may be modified toenhance complex formation, to modulate RNA nanostructure: tumor specificantigen ratios and to operably link one or more agents. In certainembodiments, the tumor-specific antigen is TKD modified to add a C atthe N-terminus. In certain embodiments, the tumor-specific antigen isTKD modified to add from 1 to 15 lysine residues at the C-terminus. Incertain embodiments, the tumor-specific antigen is TKD modified to add10 lysine residues at the C-terminus. In certain embodiments, thetumor-specific antigen is TKD modified to add a C at the N-terminus andfrom 1 to 15 lysine at the C-terminus. In certain embodiments, thetumor-specific antigen is TKD modified to add a C at the N-terminus and10 lysine at the C-terminus.

In certain embodiments, one or more of R₃ and/or R₄ can be an agent. Insome embodiments, one or more agents (e.g., 1 to 50, 2 to 50, 2 to 20,or any number of agents between the aforementioned numbers of agents)can be operably linked to the RNA nanostructure. In some embodiments,the agent can be selected from a diagnostic agents or therapeuticagents. In certain embodiments, at least one diagnostic agent isoperably linked to the RNA nanostructure. In certain embodiments, atleast one diagnostic agent is operably linked to the tumor-specificantigen. In certain embodiments, at least one therapeutic agent isoperably linked to the RNA nanostructure. In certain embodiments, atleast one therapeutic agent is operably linked to the tumor-specificantigen. In certain embodiments, at least one diagnostic agent and atleast one therapeutic agent are operably linked to the RNAnanostructure. In certain embodiments, at least one diagnostic agent andat least one therapeutic agent are operably linked to the tumor-specificantigen.

Diagnostic agents include imaging agents, e.g., fluorophores,radioisotopes, and colorimetric indicators.

As used herein, the term “therapeutic agent” includes agents thatprovide a therapeutically desirable effect when administered to subject.The agent may be of natural or synthetic origin. For example, it may bea nucleic acid, a polypeptide, a protein, a peptide, a radioisotope,saccharide or polysaccharide or an organic compound, such as a smallmolecule. The term “small molecule” includes organic molecules having amolecular weight of less than about, e.g., 1000 daltons. In oneembodiment a small molecule can have a molecular weight of less thanabout 800 daltons. In another embodiment a small molecule can have amolecular weight of less than about 500 daltons.

In certain embodiments, the therapeutic agent is an immuno-stimulatoryagent, a radioisotope, a chemotherapeutic drug (e.g., doxorubicin) or animmuno-therapy agent, such as antibody or an antibody fragment. Incertain embodiments, the therapeutic agent is a vaccine, such as acancer vaccine. In certain embodiments, the therapeutic agent is a tumortargeting agent, such as a monoclonal tumor-specific antibody or anaptamer. In certain embodiments, the therapeutic agent is an antibody orfragment thereof. The antibody or fragment thereof can be selected froma monoclonal antibody, a polyclonal antibody, an ScFv, or a nanobody. Insome embodiments, the antibody is an anti-PD1 antibody as describedherein. In certain embodiments, the therapeutic agent is an antigen(e.g., a tumor associated antigen or a tumor specific antigen). Incertain embodiments, the therapeutic agent is one or more tumor antigenpeptide(s).

In some embodiments, the therapeutic agent is a chemotherapeutic agent.As used herein, the term “chemotherapeutic agent” refers to a biological(large molecule) or chemical (small molecule) compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, proteins,antibodies, photosensitizers, and kinase inhibitors. Chemotherapeuticagents include compounds used in “targeted therapy” and non-targetedconventional chemotherapy.

In certain embodiments, the therapeutic agent is a vasoconstrictor. Incertain embodiments, the vasoconstrictor is selected from thrombin,prothrombin, rhThrombin, fragments thereof, and combinations thereof. Incertain embodiments, the therapeutic agent is thrombin. In someembodiments, the thrombin can be human thrombin, bovine thrombin, ormurine thrombin. In some embodiments, the thrombin can be thrombinalpha.

RNA Nanostructure Double-Robots

Folding nucleic acids into well-defined and compacted nanostructures,i.e., in nanometer scale (such as RNA origami described above), has beenshown to improve their nuclease resistance and lower the toxicity. Thus,RNA origami with defined structure are used as a new adjuvant platform.Nucleic acid nanostructures are excellent nano-scaffolds formolecular-cargo delivery, such as for aptamers, proteins, and drugmolecules. When conjugated with targeting compounds and molecularswitches, the RNA origami co-delivers functional cargos to the desiredlocations with a triggered and delayed release.

The design of RNA nanostructure robots as described herein combinesmultiple functional devices into a single molecular robot for cancerimmunotherapy. Moreover, RNA-based nanostructure double-robots can beproduced in a large quantity at a low cost.

In some embodiments, RNA nanostructure double-robots were designed fromsingle stranded RNA molecules (“ssRNA”) (as described above), in which atumor-targeting molecule, e.g., nucleolin-binding aptamer, is attachedto the double-robot to allow this robot device targeted to the tumormicroenvironment. In addition, the double-robot contains two functionalmodules, “NR₁” (RNA nanorobot-1) and “NR₂” (RNA nanorobot-2), which arelinked by one or more linker L. In some aspects, the linker L istubable. In some aspects, the tunable properties of the linker L arepH-responsiveness, where the linker is responsive to the relatively lowpH in the tumor microenvironment. As defined herein, “low pH” is a pHbelow about 7, such as below about 6, below about 5, below about 4,below about 3 or below about 2. One module, NR₁, function as bothimmune-adjuvant and RNA nanostructure scaffold for antigen specificdelivery and induction of antigen-specific immunity. In someembodiments, the other module, NR₂, deliver a first species of R₄, whichis a functional cargo molecule. In some embodiments, the functionalcargo molecule is a checkpoint inhibitor antagonist. In someembodiments, the checkpoint inhibitor antagonistarein a form ofantibodies, aptamers, or drugs. The opening of this module is controlledby a second species of R₄, which can be an aptamer that is specificallybinding to Interferon gamma (IFN-gamma) that is usually produced byactivated T cells. However, local accumulation of IFN-gamma negativelyregulates T cell function by upregulating PD1 expression on theseactivated T cells (Ribas, A. 2015. Adaptive Immune Resistance: Howcancer protects from immune Attack. Cancer Discovery. 5: 914-919). Thus,the delayed release of anti-PD1 antibodies or aptamers from theRNA-nanocage robot in response to IFN-gamma effectively counteracts thecheckpoint action without compromising the generation of tumor-specificimmunity.

An RNA nanostructure robot was constructed with multiple functions usingsingle-stranded RNA origami technology. In the first RNA nanorobot, NR₁,the RNA origami was loaded with tumor specific peptide antigens and wastaken up by antigen presenting cells (APCs) upon intraperitonealinjection. This elicits strong anti-tumor immunity and eventually killscancer cells. Certain embodiments provide a method of inducing an immuneresponse in a subject (e.g., a mammal, such as a human), comprisingadministering to the subject an effective amount of an RNA nanostructurerobot, complex or composition as described herein. Certain embodimentsprovide a method of enhancing/increasing pro-inflammatory cytokines in asubject (e.g., a mammal, such as a human), comprising administering tothe subject a therapeutically effective amount of an RNA nanostructurerobot, complex or composition as described herein. Certain embodimentsprovide a method of activating immune cells by specific triggering ofTLR3 signaling pathway in a subject (e.g., a mammal, such as a human),comprising administering to the subject a therapeutically effectiveamount of an RNA nanostructure robot, complex or composition asdescribed herein. In some embodiments, the second RNA nanobobot, NR₂,comprises an RNA origami cage loaded with checkpoint inhibitors andlocked by IFN-gamma aptamers. The RNA origami cage contains nucleolinaptamers that direct the specific delivery to tumor site. Upon IFN-gammarelease from T-cells, the RNA origami cage unlocks and releases thecheckpoint inhibitors (either anti-PD1 antibodies or aptamers) tomitigate immunosuppression in the tumor microenvironment. Thesingle-stranded RNA origami structures are designed to deliversaptamers, antibodies and drugs into tumor environment with a triggeredand delayed release.

The immune system plays an important role in the development of a cancercell from a normal cell. Thus, in some embodiments, the inventionfeatures a method comprising a RNA nanostructure comprising an agentthat targets one or more immune system checkpoints. Immune systemcheckpoints are inhibitory signaling pathways modulated by checkpointproteins that turn off immune system effectors cells, for example, Tcells. Some embodiments of the invention can include, checkpointinhibitor antagonists (also referred to as “immune-checkpoint inhibitorsantagonists”), which target the checkpoint proteins. The targetcheckpoint proteins include, but are not limited to, indoleamine (2,3)-dioxygenase (IDO); programmed cytotoxic T-lymphocyte antigen 4(CTLA-4), programmed death-1 (PD-1); programmed death-ligand 1 (PD-L1);PD-L2; lymphocyte activation gene 3 (LAG3); and B7 homolog 3 (B7-H3).

In some embodiments, the checkpoint inhibitor antagonists bind toligands or proteins that are found on any of the family of T cellregulators, such as CD28/CTLA-4. Targets of checkpoint inhibitorantagonists include, but are not limited to, receptors or co-receptors(e.g., CTLA-4; CD8) expressed on immune system effector or regulatorcells (e.g., T cells); proteins expressed on the surface ofantigen-presenting cells (i.e., expressed on the surface of activated Tcells, including PD-1, PD-2, PD-L1 and PD-L2); metabolic enzymes ormetabolic enzymes that are expressed by both tumor andtumor-infiltrating cells (e.g., indoleamine (IDO), including isoforms,such as IDO1 and IDO2); proteins that belong to the immunoglobulinsuperfamily (e.g., lymphocyte-activation gene 3, also referred to asLAG3); proteins that belong to the B7 superfamily (e.g., B7-H3 orhomologs thereof;). B7 proteins are found on both activated antigenpresenting cells and T cells.

Checkpoint inhibitor antagonists useful in the present invention includebut are not limited to: Ipilimumab (Yervoy), Tremelimumab (formerlyticilimumab), Pidilizumab (CT-011), Nivolumab (BMS-936558),Lambrolizumab (MK-3475), MPDL3280A, BMS-936559, AMP-224, IMP321(ImmuFact), MGA271, Indoximod, INCB024360, IMP321 (Immuntep®),BMS-986016, LAG525, MBG453, CA-170, JNJ-61610588, Endoblituzumab(MGA271), MGD009, 8H9, Lirilumab, IPH4102, CPI-444, MEDI9447, OMP-31M32,Trabedersen (AP12009), M7824, Galusertinib (LY2157299), IPI-549,Hu5F9-G4, TTI-621 (SIRPalphaFc), 9B12, MOXR 0916, PF08600, MED6383,MED10562, INCAGN01949, GSK3174998, TRX-518, BMS-986156, AMG 228,MEDI1873, MEDI6469, MK-4166, INCAGN01876, GWN323, JTX-2011, GSK3359609,MEDI-570, Utomilumab, Urelumab, ARGX-110, BMS-936561, MDX-1203,Varilumab, CP-870893, APX005M, ADC-1013, JNJ-64457107, SEA-CD40,R07009789, BMX-986205, Indoximod, Epacadostat, MEDI9197, pixatimod,NKTR-214, CB-1158, LTX-315, and AM0010.

The RNA nanostructure robots (linked double robots or single chaindouble robots) described herein offer unprecedented multi-functionalcapabilities, including 1) tumor-targeted delivery; 2) potent adjuvantplatform for construction tumor-specific vaccines; and 3) programmablerelease of checkpoint inhibitors. In some embodiments, the programmablerelease of checkpoint inhibitors occurs by the inclusion of a RNAnanostructure which is in the form of a locked cage with internalizedcargo compounds which opens in the presence of a targeted stimulus.Thus, this novel RNA-robot functions at both directions of the immunereactions, i.e., activating tumor-specific immunity as well asantagonizing tumor-associated immunosuppression, which should helpsynergize and maximize anti-tumor immunity.

Without being bound by theory, the RNA nanostructure robots activatetumor-specific immunity by presenting a tumor antigen peptide toeffector T-cells on an immune cell. Effector T cells (also calledregulatory T cells, or Treg cells, or suppressor T cells) are immunesystem cells that inhibit a T cell response and are important in leadingto cell-mediated immunity. Effector T cells contain cell surface markersincluding CD25 (the IL-2 receptor), CD45RB, CTLA4 and FOXP3. A T cellresponse is an immune-type response that mediated through T cells. WhenT cells are activated, they will divide and generate more T cells (e.g.Th1 or Th2 cells, also called CD4+ cells or T helper cells; memory Tcells; or cytotoxic T cells; also referred to as CTLs or CD8+ T cells).Once activated, T helper cells divide and generate cytokines thatregulate or otherwise assist other immune system cells to elicit animmune system response. Memory T cells also generate additional effectorT cells upon activation after previous exposure to an antigen. CytotoxicT cells kill tumor cells when presented with a target that theyrecognize.

Immuno-Stimulatory RNA Nanostructure Robot

Certain embodiments of the invention provide an RNA nanostructure havingimmuno-stimulatory properties. As used herein, an immune-stimulatory RNAnanostructure stimulates the immune system by, for example, inducingactivation or increasing activity of any components of the immunesystem, or by reducing inhibition of the immune system. In certainembodiments, the RNA nanostructure is an agonist of a patternrecognition receptor. As used herein, the terms “pattern recognitionreceptor” or “PRR” refer to proteins expressed by cells of the innateimmune system, such as dendritic cells, macrophages, monocytes,neutrophils and epithelial cells, to identify two classes of molecules:pathogen-associated molecular patterns (PAMPs), which are associatedwith microbial pathogens, and damage-associated molecular patterns(DAMPs), which are associated with components of host's cells that arereleased during cell damage or death. PRRs also mediate the initiationof antigen-specific adaptive immune response and release of inflammatorycytokines. In certain embodiments, the PRR is a toll-like receptor (TLR)(e.g., TLR3 or TLR7).

Accordingly, certain embodiments provide an RNA nanostructure that isimmuno-stimulatory. As used herein, an immuno-stimulatory RNAnanostructure stimulates the immune system thereby inducing activationor increasing activity of any components of the immune system. In someembodiments, the immune-stimulatory RNA structures described hereinstimulate immune cell activation, boost anti-tumor immunity, increaseanti-tumor (pro-inflammatory) cytokines and/or reduce immunosuppressivecytokines. For example, in some embodiments immuno-stimulatory RNAstructures described herein: activate immune cells, e.g., T helpercells, T cells (including CD69+ activated T cells), dendritic cells,natural killer cells, macrophages, reprogram the cytokinemicroenvironment by, for example, decreasing levels of immunosuppressivecytokines e.g., TGF beta (TGFβ1, TGFβ2, IL10, and IL4 and/or increasingproduction of anti-tumor (pro-inflammatory) cytokines, for example,interferon gamma and TNF-alpha; inhibit or suppress tumor growth, causetumor regression and/or induce tumor immunity; stimulate splenic B and Tcells; or activate the TLR3-signaling pathway.

Stability

Antigen peptides in contact with the RNA nanostructures via apolylysine-RNA interaction exhibit enhanced stability over that ofpolylysine-polyIC complexes (FIG. 37). The enhanced stability of thepolylysine-RNA nanostructures demonstrates that the RNA nanostructurescirculate longer in the circulatory system after administration,resulting in a higher potency.

Nuclease Resistance

In certain embodiments, the RNA nanostructure robot has increasednuclease resistance (e.g., as compared to a control, such as an unfoldedssRNA molecule comprising the same nucleic acid sequence as the RNAnanostructure). In certain embodiments, nuclease resistance of the RNAnanostructure is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or more than a control. The increased nuclease resistance enables theRNA nanostructure robot to circulate in the body longer afteradministration of a dose, resulting in higher potency of the dose.

Linkages

In some embodiments, the linkage between NR₁ to L, NR₂ to L, NR₁ to R₃,or NR₂ to R₄ is any group that can connect the NR₁ or NR₂ RNAnanostructure robots to each other or the moieties R₃ and/or R₄ usingchemical methods, provided that the connection method does not interferewith the function of the R₃ and/or R₄ moieties or the RNA nanostructure.Chemistries that can be used to link the R₃ and/or R₄ moieties to anoligonucleotide can include or exclude: disulfide linkages, aminolinkages, covalent linkages, etc. In certain embodiments, aliphatic orethylene glycol linkers can be used. In certain embodimentsphosphodiester, phosphorothioate and/or other modified linkages areused. In certain embodiments, the linker is a binding pair. In certainembodiments, the “binding pair” refers to two molecules which interactwith each other through any of a variety of molecular forces including,for example, ionic, covalent, hydrophobic, van der Waals, and hydrogenbonding, so that the pair have the property of binding specifically toeach other. Specific binding means that the binding pair members exhibitbinding to each other under conditions where they do not bind to anothermolecule. Examples of binding pairs are biotin-avidin, hormone-receptor,receptor-ligand, enzyme-substrate probe, IgG-protein A,antigen-antibody, aptamer-target and the like. In certain embodiments, afirst member of the binding pair comprises avidin or streptavidin and asecond member of the binding pair comprises biotin.

In some embodiments, the R₃ and/or R₄ moieties can comprise a loadingoligonucleotide. In some embodiments, the loading oligonucleotide isdesigned to have all or a portion of the oligonucleotide sequence to becomplementary to all or a portion of the ssRNA of NR₁ and/or NR₂. Insome embodiments, the oligonucleotide sequence can comprise RNA, DNA,modified RNA, modified DNA, or combinations thereof. In someembodiments, the 5′- or 3′-ends of the oligonucleotide is modified witha oligonucleoside which can bind to a therapeutic and/or diagnosticagent. In some embodiments, the oligonucleotide is 5′-modified with athiol-comprising nucleotide. In some embodiments, the thiol-comprisingnucleotide is further reacted with a cross-functional linker. In someembodiments, the cross-functional linker is sulfo-SMCC((sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate),ThermoFisher cat. 22322). In some embodiments, the cross-functionallinker is Sulfo-LC-SPDP (sulfosuccinimidyl6-(3′-(2-pyridyldithio)propionamido)hexanoate) (ThermoFisher cat.21650). In some embodiments, the sequence of the loading oligonucleotideis selected from the following loading oligonucleotide sequences:

TH-43 (SEQ ID NO: 124) AAAAAAAAAAAAAAACAAAAATCATTGCTCCTTTTGATAAGTTTCAT;TH-44 (SEQ ID NO: 125) AAAAAAAAAAAAAAAAAAGATTCAGGGGGTAATAGTAAACCATAAAT;TH-57 (SEQ ID NO: 39) AAAAAAAAAAAAAAATTTGCCAGATCAGTTGAGATTTAGTGGTTTAA;TH-64 (SEQ ID NO: 40) AAAAAAAAAAAAAAAGCAAATATCGCGTCTGGCCTTCCTGGCCTCAG;TH-65 (SEQ ID NO: 41) AAAAAAAAAAAAAAATATATTTTAGCTGATAAATTAATGTTGTATAA;TH-78 (SEQ ID NO: 42) AAAAAAAAAAAAAAAACCGTTCTAAATGCAATGCCTGAGAGGTGGCA;TH-139 (SEQ ID NO: 43) AAAAAAAAAAAAAAAATTATTTAACCCAGCTACAATTTTCAAGAACG;TH-140 (SEQ ID NO: 44) AAAAAAAAAAAAAAGAAGGAAAATAAGAGCAAGAAACAACAGCCAT;TH-153 (SEQ ID NO: 45) AAAAAAAAAAAAAAAGCCCAATACCGAGGAAACGCAATAGGTTTACC;TH-160 (SEQ ID NO: 46) AAAAAAAAAAAAAAATAACCTCCATATGTGAGTGAATAAACAAAATC;TH-161 (SEQ ID NO: 47) AAAAAAAAAAAAAAACATATTTAGAAATACCGACCGTGTTACCTTTT;or TH-174 (SEQ ID NO: 48)AAAAAAAAAAAAAAAAATGGTTTACAACGCCAACATGTAGTTCAGCT.Any of the aforementioned oligonucleotide sequences is 5′ or 3′ modifiedas described herein.

In some embodiments, the cross-functional compound can further bereacted with an agent. In some aspects, the agent is a protein orpeptide comprising a lysine amino acid. The amine on the lysine canreact with the amino-reactive cross-functional compound to form aloading oligonucleotide-functionalized agent. In some embodiments, theoligonucleotide-functionalized agent is hybridized to a portion or allof the ssRNA of NR₁ and/or NR₂.

In some embodiments, this invention provides for a RNA nanostructurerobot, wherein the ssRNA sequence comprises a modified ribonucleic acid.The ribonucleic acid can comprise an alkyl amine functional group. Insome embodiments, the amino-modified ribonucleoside is incorporated intothe ssRNA sequence by the addition of5-Aminoallyluridine-5′-Triphosphate during a transcription step offorming the ssRNA sequence.

In some embodiments, this invention provides for a RNA nanostructurerobot, wherein wherein the therapeutic agent is a protein. The proteinis selected from thrombin, prothrombin, or mixtures thereof. In someembodiments, the protein or peptide is conjugated to the amino-modifiedribonucleotide by means of a cross-functional compound as describedherein. In some embodiments, the maleimide group on the sulfo-SMCC canreact to a cysteine on a non-reduced or reduced form of the thrombinmolecule, and the sulfosuccinimidyl group on the sulfo-SMCC can react tothe amino-modified ribonucleoside. In some embodiments, the maleimidegroup on the sulfo-SMCC can react to a loading oligonucleotide which hasbeen modified to include a thiol-group to form a loadingoligonucleotide-functionalized agent.

Compositions and Kits

Certain embodiments of the invention also provide a compositioncomprising an RNA nanostructure complex described herein and a carrier.In certain embodiments, the composition comprises a plurality of RNAnanostructures and a carrier.

In certain embodiments, the composition further comprises at least onetherapeutic agent as described herein.

In certain embodiments, the composition is a pharmaceutical compositionand the carrier is a pharmaceutically acceptable carrier.

Certain embodiments of the invention also provide a vaccine comprisingan RNA nanostructure complex as described herein.

The present invention further provides kits for practicing the presentmethods.

Accordingly, certain embodiments of the invention provide a kitcomprising an RNA nanostructure complex described herein andinstructions for administering the RNA nanostructure to induce an immuneresponse (e.g., anti-tumor immunity) or to treat a disease or condition.In certain embodiments, the kit further comprises a therapeutic agentdescribed herein and instructions for administering the therapeuticagent in combination (e.g., simultaneously or sequentially) with the RNAnanostructure complex.

Certain Methods

An RNA nanostructure complex described herein may be used as animmune-adjuvant to boost an immune response (e.g., inducing anti-tumorimmunity).

Accordingly, certain embodiments of the invention provide a method ofinducing an immune response in a subject (e.g., a mammal, such as ahuman), comprising administering to the subject an effective amount ofan RNA nanostructure complex or composition as described herein. Incertain embodiments, the administration increases an immune response byat least about, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more(e.g., as compared to a control). Methods of measuring an immuneresponse include using an assay described in the Example. The phrase“inducing an immune response” refers to the activation of an immunecell. Methods of measuring an immune response include using an assaydescribed in the Example. The phrase “effective amount” means an amountof an RNA nanostructure described herein that induces an immuneresponse.

Certain embodiments of the invention also provide a method of treating adisease or disorder in a subject, comprising administering to thesubject a therapeutically effective amount of an RNA nanostructurecomplex or a composition as described herein.

In certain embodiments, a method of the invention further comprisesadministering at least one therapeutic agent to the subject.

The at least one therapeutic agent may be administered in combinationwith the RNA nanostructure. As used herein, the phrase “in combination”refers to the simultaneous or sequential administration of the RNAnanostructure and the at least one therapeutic agent. For simultaneousadministration, the RNA nanostructure and the at least one therapeuticagent may be present in a single composition or may be separate (e.g.,may be administered by the same or different routes).

Certain embodiments of the invention provide a RNA nanostructure complexor a composition as described herein for use in medical therapy.

Certain embodiments of the invention provide the use of an RNAnanostructure complex or a composition as described herein for themanufacture of a medicament for inducing an immune response in a subject(e.g., a mammal, such as a human).

Certain embodiments of the invention provide the use of an RNAnanostructure complex or a composition as described herein for themanufacture of a medicament for inducing an immune response in a subject(e.g., a mammal, such as a human), in combination with at least onetherapeutic agent.

Certain embodiments of the invention provide an RNA nanostructurecomplex or a composition as described herein for inducing an immuneresponse.

Certain embodiments of the invention provide an RNA nanostructurecomplex or a composition as described herein for inducing an immuneresponse, in combination with at least one therapeutic agent.

Certain embodiments of the invention provide the use of an RNAnanostructure complex or a composition as described herein for themanufacture of a medicament for treating a disease or disorder in asubject.

Certain embodiments of the invention provide the use of an RNAnanostructure complex or a composition as described herein for themanufacture of a medicament for treating a disease or disorder in asubject, in combination with at least one therapeutic agent.

Certain embodiments of the invention provide an RNA nanostructurecomplex or a composition as described herein for the prophylactic ortherapeutic treatment a disease or disorder.

Certain embodiments of the invention provide an RNA nanostructurecomplex or a composition as described herein for the prophylactic ortherapeutic treatment of a disease or disorder, in combination with atleast one therapeutic agent.

In certain embodiments, the disease or disorder is a condition thatrequires a boost of the host immunity. In certain embodiments, thedisease or disorder is a hyperproliferative disorder, such as cancer. Incertain embodiments, the disease or disorder is an infectious disease.

In certain embodiments, the cancer is carcinoma, lymphoma, blastoma,sarcoma, or leukemia. In certain embodiments, the cancer is a solidtumor cancer.

In certain embodiments, the cancer is squamous cell cancer, small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung,squamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, renal cell carcinoma, gastrointestinal cancer, gastric cancer,esophageal cancer, pancreatic cancer, glioma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g.,endocrine resistant breast cancer), colon cancer, rectal cancer, lungcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma, melanoma, leukemia, or head and neck cancer.In certain embodiments, the cancer is breast cancer.

In certain embodiments, the therapeutic agent is a therapeutic agentdescribed herein. For example, in certain embodiments, the therapeuticagent is an immuno-stimulatory agent, a radioisotope, a chemotherapeuticdrug (e.g., doxorubicin) or an immuno-therapy agent, such as antibody oran antibody fragment. In certain embodiments, the therapeutic agent is avaccine, such as a cancer vaccine. In certain embodiments, thetherapeutic agent is a tumor targeting agent, such as a monoclonaltumor-specific antibody or an aptamer. In certain embodiments, thetherapeutic agent is an antibody (e.g., a monoclonal antibody, e.g., ananti-PD1 antibody). In certain embodiments, the therapeutic agent is anantigen (e.g., a tumor associated antigen or a tumor specific antigen).In certain embodiments, the therapeutic agent is a tumor antigenpeptide(s).

Administration

In some embodiments, methods of the invention comprise administering aRNA nanostructure described herein, and optionally, a therapeutic agentto a subject. Such compounds (i.e., a RNA nanostructure and/ortherapeutic agent) may be formulated as a pharmaceutical composition andadministered to a mammalian host, such as a human patient in a varietyof forms adapted to the chosen route of administration, i.e., orally orparenterally, by intravenous, intramuscular, intraperitoneal or topicalor subcutaneous routes.

The active compound may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts is prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle isa solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity ismaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms is brought about by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions is broughtabout by the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated using one or more physiologically acceptablecarriers or excipients. Any suitable concentration of the RNAnanostructure robot may be used, and any active pharmaceuticalingredient will be administered in an amount effective to achieve itsintended purpose.

A variety of suspending fluids or carriers may be employed to suspendthe RNA nanostructure robot composition. Such fluids include withoutlimitation: sterile water, saline, buffer, or complex fluids derivedfrom growth medium or other biological fluids. Preservatives,stabilizers and antibiotics may be employed in the RNA nanostructurerobot composition.

Methods of making a pharmaceutical composition include admixing at leastone active compound or agent, as defined above, together with one ormore other pharmaceutically acceptable ingredients, such as carriers,diluents, excipients, and the like. When formulated as discrete units,such as tablets or capsule or suspension, each unit contains apredetermined amount of the active compound or agent.

Suitable formulations will depend on the method of administration. Thepharmaceutical composition is preferably administered by intradermaladministration, but other routes of administration include for exampleoral, buccal, rectal, parenteral, intramuscular, subcutaneous,intraperitoneal, transdermal, intrathecal, nasal, intracheal. Thepolyvalent vaccine can also be administered to the lymph nodes such asaxillary, inguinal or cervial lymph nodes. The active agent may besystemic after administration or may be localized by the use of regionaladministration, intramural administration, or use of an implant thatacts to retain the active dose at the site of implantation.

Pharmaceutical compositions described herein may be administereddirectly, they may also be formulated to include at least onepharmaceutically-acceptable, nontoxic carriers of diluents, adjuvants,or non-toxic, nontherapeutic, fillers, buffers, preservatives,lubricants, solubilizers, surfactants, wetting agents, masking agents,and coloring agents. Also, as described herein, such formulation mayalso include other active agents, for example, other therapeutic orprophylactic agents, nonimmunogenic stabilizers, excipients and thelike. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents and detergents.

Adjuvants can include or exclude: polymers, copolymers such aspolyoxyethylene-polyoxypropylene co-polymers, including blockco-polymers; polymer P1005; monotide ISA72; Freund's incompleteadjuvant; sorbitan monooleate; squalene; CRL-8300 adjuvant; alum; QS 21,muramyl dipeptide; trehalose; bacterial extracts, includingmycobacterial extracts; detoxified endotoxins; membrane lipids;water-in-oil mixtures, water-in-oil-in-water mixtures or combinationsthereof.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds is dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents isadded to optimize the properties for a given use. The resultant liquidcompositions is applied from absorbent pads, used to impregnate bandagesand other dressings, or sprayed onto the affected area using pump-typeor aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Dermatological compositions which can be used to deliver a compound tothe skin are can include or exclude, for example, see Jacquet et al.(U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al.(U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508), eachof which is incorporated by reference herein in their entirety.

Useful dosages of compounds can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949, hereinincorporated by reference.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

The compound may be conveniently formulated in unit dosage form. In oneembodiment, the invention provides a composition comprising a compoundformulated in such a unit dosage form. The desired dose may convenientlybe presented in a single dose or as divided doses administered atappropriate intervals, for example, as two, three, four or moresub-doses per day. The sub-dose itself may be further divided, e.g.,into a number of discrete loosely spaced administrations; such asmultiple inhalations from an insufflator or by application of aplurality of drops into the eye.

Certain Definitions

As used herein, the term “about” means±10%.

“Operably-linked” refers to the association two chemical moieties sothat the function of one is affected by the other, e.g., an arrangementof elements wherein the components so described are configured so as toperform their usual function.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, made of monomers (nucleotides) containing a sugar,phosphate and a base that is either a purine or pyrimidine. Unlessspecifically limited, the term encompasses nucleic acids containingsynthetic analogs of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also encompasses conservativelymodified variants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues.

The terms “nucleotide sequence”, “polynucleic acid”, or “nucleic acidsequence” refer to a sequence of bases (purines and/or pyrimidines) in apolymer of DNA or RNA, which can be single-stranded or double-stranded,optionally containing synthetic, non-natural or altered nucleotide basescapable of incorporation into DNA or RNA polymers, and/or backbonemodifications (e.g., a modified oligomer, such as a morpholino oligomer,phosphorodiamate morpholino oligomer or vivo-mopholino). The terms“oligo”, “oligonucleotide” and “oligomer” may be used interchangeablyand refer to such sequences of purines and/or pyrimidines. The terms“modified oligos”, “modified oligonucleotides”, “modified oligomers”,“modified ribonucleosides” or “modified ribonucleotides” may besimilarly used interchangeably, and refer to such sequences that containsynthetic, non-natural or altered bases and/or backbone modifications.

In some embodiments, the modified oligos can comprise chemicalmodifications to the internucleotide phosphate linkages and/or to thebackbone sugar.

Modified nucleotides can include or exclude: alkylated purines and/orpyrimidines; acylated purines and/or pyrimidines; or other heterocycles.These classes of pyrimidines and purines can include or exclude:pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine;4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil;5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil;5-carboxymethylaminomethyl uracil; dihydrouracil; inosine;N6-isopentyl-adenine; 1-methyladenine; 1-methylpseudouracil;1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine;3-methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine;5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil;3-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil;2-methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methylester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil;4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester;uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil;5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil;5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine;methylpsuedouracil; 1-methylguanine; 1-methylcytosine. Backbonemodifications are can include or exclude: chemical modifications to thephosphate linkage (e.g., phosphorodiamidate, phosphorothioate (PS),N3′phosphoramidate (NP), boranophosphate, 2′,5′phosphodiester,amide-linked, phosphonoacetate (PACE), morpholino, peptide nucleic acid(PNA) and inverted linkages (5′-5′ and 3′-3′ linkages)) and sugarmodifications (e.g., 2′-O-Me, UNA, LNA).

The oligonucleotides described herein may be synthesized using standardsolid or solution phase synthesis methods. In certain embodiments, theoligonucleotides are synthesized using solid-phase phosphoramiditechemistry (U.S. Pat. No. 6,773,885) with automated synthesizers.Chemical synthesis of nucleic acids allows for the production of variousforms of the nucleic acids with modified linkages, chimericcompositions, and nonstandard bases or modifying groups attached inchosen places through the nucleic acid's entire length.

Certain embodiments of the invention encompass isolated or substantiallypurified nucleic acid compositions. In the context of the presentinvention, an “isolated” or “purified” DNA molecule or RNA molecule is aDNA molecule or RNA molecule that exists apart from its nativeenvironment and is therefore not a product of nature. An isolated DNAmolecule or RNA molecule may exist in a purified form or may exist in anon-native environment such as, for example, a transgenic host cell. Forexample, an “isolated” or “purified” nucleic acid molecule issubstantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In oneembodiment, an “isolated” nucleic acid is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived.

By “portion” or “fragment,” as it relates to a nucleic acid molecule,sequence or segment of the invention, when it is linked to othersequences for expression, is meant a sequence having at least 80nucleotides, at least 150 nucleotides, or at least 400 nucleotides. Ifnot employed for expressing, a “portion” or “fragment” means at least 9,at least 12, at least 15, or at least 20, consecutive nucleotides, e.g.,probes and primers (oligonucleotides), corresponding to the nucleotidesequence of the nucleic acid molecules of the invention.

“Recombinant DNA molecule” is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (3rd edition, 2001).

“Homology” refers to the percent identity between two polynucleotides ortwo polypeptide sequences. Two DNA or polypeptide sequences are“homologous” to each other when the sequences exhibit at least about 75%to 85% (including 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and85%), at least about 90%, or at least about 95% to 99% (including 95%,96%, 97%, 98%, 99%) contiguous sequence identity over a defined lengthof the sequences.

The following terms are used to describe the sequence relationshipsbetween two or more nucleotide sequences: “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” “substantial identity,” and “complementarity”.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally is 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison can be performed. Thus,the determination of percent identity, including sequencecomplementarity, between any two sequences is accomplished using amathematical algorithm. Non-limiting examples of such mathematicalalgorithms are the algorithm of Myers and Miller (Myers and Miller,CABIOS, 4, 11 (1988)); the local homology algorithm of Smith et al.(Smith et al., Adv. Appl. Math., 2, 482 (1981)); the homology alignmentalgorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443(1970)); the search-for-similarity-method of Pearson and Lipman (Pearsonand Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444 (1988)); the algorithmof Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA,87, 2264 (1990)), modified as in Karlin and Altschul (Karlin andAltschul, Proc. Natl. Acad. Sci. USA 90, 5873 (1993)).

Computer implementations of these mathematical algorithms is utilizedfor comparison of sequences to determine sequence identity orcomplementarity. Such implementations include, but are not limited to:CLUSTAL in the PC/Gene program (available from Intelligenetics, MountainView, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8(available from Genetics Computer Group (GCG), 575. Science Drive,Madison, Wis., USA). Alignments using these programs is performed usingthe default parameters. The CLUSTAL program is well described by Higginset al. (Higgins et al., CABIOS, 5, 151 (1989)); Corpet et al. (Corpet etal., Nucl. Acids Res., 16, 10881 (1988)); Huang et al. (Huang et al.,CABIOS, 8, 155 (1992)); and Pearson et al. (Pearson et al., Meth. Mol.Biol., 24, 307 (1994)). The ALIGN program is based on the algorithm ofMyers and Miller, supra. The BLAST programs of Altschul et al. (Altschulet al., JMB, 215, 403 (1990)) are based on the algorithm of Karlin andAltschul supra.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score is increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when thecumulative alignment score falls off by the quantity X from its maximumachieved value, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, less than about0.01, or even less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) is utilized. Alternatively, PSI-BLAST (in BLAST 2.0) is usedto perform an iterated search that detects distant relationships betweenmolecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the defaultparameters of the respective programs (e.g., BLASTN for nucleotidesequences, BLASTX for proteins) is used. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix. Alignment may also be performed manually byinspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity may be madeusing the BlastN program (version 1.4.7 or later) with its defaultparameters or any equivalent program. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide or aminoacid residue matches and an identical percent sequence identity whencompared to the corresponding alignment generated by the program.

As used herein, the terms “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference toa specified percentage of residues in the two sequences that are thesame when aligned for maximum correspondence over a specified comparisonwindow, as measured by sequence comparison algorithms or by visualinspection.

As used herein, the term, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, the term “identity” or “substantial identity” ofpolynucleotide sequences means that a polynucleotide comprises asequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequenceidentity, compared to a reference sequence using one of the alignmentprograms described using standard parameters.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. “Bind(s)substantially” refers to complementary hybridization between a probenucleic acid and a target nucleic acid and embraces minor mismatchesthat is accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence.

The term “complementary” as used herein refers to the broad concept ofcomplementary base pairing between two nucleic acids aligned in anantisense position in relation to each other. When a nucleotide positionin both of the molecules is occupied by nucleotides normally capable ofbase pairing with each other, then the nucleic acids are considered tobe complementary to each other at this position. Thus, two nucleic acidsare substantially complementary to each other when at least about 50%,at least about 60%, or at least about 80% of corresponding positions ineach of the molecules are occupied by nucleotides which normally basepair with each other (e.g., A:T (A:U for RNA) and G:C nucleotide pairs).

As used herein, the term “derived” or “directed to” with respect to anucleotide molecule means that the molecule has complementary sequenceidentity to a particular molecule of interest.

As used herein, the term “therapeutically effective amount” means anamount of a compound of the present invention that (i) treats theparticular disease, condition, or disorder, (ii) attenuates,ameliorates, or eliminates one or more symptoms of the particulardisease, condition, or disorder, or (iii) prevents or delays the onsetof one or more symptoms of the particular disease, condition, ordisorder described herein. In the case of cancer, the therapeuticallyeffective amount of the drug may reduce the number of cancer cells;reduce the tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the cancer. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy can be measured, forexample, by assessing the time to disease progression (TTP) and/ordetermining the response rate (RR).

As used herein, the term “subject” as used herein refers to humans,higher non-human primates, rodents, domestic, cows, horses, pigs, sheep,dogs and cats. In one embodiment, the subject is a human.

As used herein, the terms “treat” and “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or decrease an undesired physiological changeor disorder. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer. Gastriccancer, as used herein, includes stomach cancer, which can develop inany part of the stomach and may spread throughout the stomach and toother organs; particularly the esophagus, lungs, lymph nodes, and theliver.

As used herein, the term “synergistic” as used herein refers to atherapeutic combination that is more effective than the additive effectsof the two or more single agents. A determination of a synergisticinteraction between an RNA-origami and one or more chemotherapeuticagent may be based on the results obtained from the assays describedherein. The results of these assays can be analyzed using the Chou andTalalay combination method and Dose-Effect Analysis with CalcuSynsoftware in order to obtain a Combination Index (Chou and Talalay, 1984,Adv. Enzyme Regul. 22:27-55). The combinations provided by thisinvention can be evaluated in several assay systems, and the data can beanalyzed utilizing a standard program for quantifying synergism,additivism, and antagonism among anticancer agents. The programutilized, for example in FIG. 12, is that described by Chou and Talalay,in “New Avenues in Developmental Cancer Chemotherapy,” Academic Press,1987, Chapter 2. Combination Index values less than 0.8 indicatessynergy, values greater than 1.2 indicate antagonism and values between0.8 to 1.2 indicate additive effects. The combination therapy mayprovide “synergy” and prove “synergistic”, i.e., the effect achievedwhen the active ingredients used together is greater than the sum of theeffects that results from using the compounds separately. A synergisticeffect may be attained when the active ingredients are: (1)co-formulated and administered or delivered simultaneously in acombined, unit dosage formulation; (2) delivered by alternation or inparallel as separate formulations; or (3) by some other regimen. Whendelivered in alternation therapy, a synergistic effect may be attainedwhen the compounds are administered or delivered sequentially, e.g., bydifferent injections in separate syringes. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together. In some examples, Combination effects wereevaluated using both the BLISS independence model and the highest singleagent (HSA) model (Lehir et al. 2007, Molecular Systems Biology 3:80).BLISS scores quantify degree of potentiation from single agents and aBLISS score >0 suggests greater than simple additivity. An HSA score >0suggests a combination effect greater than the maximum of the singleagent responses at corresponding concentrations.

Response Evaluation Criteria in Solid Tumors, Version 1.1 (RECIST v1.1),can be used to evaluate tumor responses in certain human clinicaltrials. This section provides the definitions of the criteria used todetermine objective tumor response for target lesions. “Completeresponse” (CR) is used to mean disappearance of all observable targetlesions with pathological lymph nodes (whether target or non-target)having reduction in short axis to less than about 10 mm. “Partialresponse” (PR) is used to mean at least about a 30% decrease in the sumof diameters of target lesions, taking as reference the baseline sum ofdiameters. “Progressive disease” (PD) is used to mean at least about a20% increase in the sum of diameters of target lesions, taking asreference the smallest sum on study (nadir), including baseline. Inaddition to the relative increase of about 20%, the sum alsodemonstrates an absolute increase of at least about 5 mm. In oneexample, the appearance of one or more new lesions is considered PD.“Stable disease” (SD) is used to mean neither sufficient shrinkage toqualify for PR nor sufficient increase to qualify for PD, taking asreference the smallest sum on study.

Adverse Event Grading (Severity) Scale is used to evaluate safety andtolerability with Grade 1 is mild (intervention not indicated), Grade 2is moderate (minimal, local, or noninvasive intervention indicated),Grade 3 is severe (severe or medically significant but not immediatelylife threatening; hospitalization or prolongation of hospitalizationindicated), Grade 4 is very severe, life threatening or disabling,urgent intervention indicated, and Grade 5 is death related to theadverse event.

EMBODIMENTS OF THE INVENTION

In some embodiments, the present invention provides a RNA nanostructurerobot having the sequence of (R₃)_(n)—NR₁-L-NR₂—(R₄)_(m), wherein:

-   -   NR₁ represents a first nano-robot comprising a single stranded        RNA (ssRNA) of about 1500 to 10,000 bases in length that        self-assembles into a first scaffold;    -   NR₂ represents a second nano-robot comprising a ssRNA of about        1500 to 10,000 bases in length that self-assembles into a second        scaffold;    -   L is a linker which operably links NR₁ to NR₂;    -   wherein R₃ and R₄ are independently selected from a pair of        fastener strands, an aptamer, a cargo molecule, a capture        strand, a targeting strand, or H;    -   n is an integer from 0 to 20; and    -   m is an integer from 0 to 20.

In certain embodiments, the linker L is an oligonucleotide. Theoligonucleotide is comprised of DNA, RNA, modified DNA, modified RNA, orcombinations thereof.

In certain embodiments, the RNA nanostructure robot is a single motif,where the sequence NR₁-L-NR₂ is continuous. The NR₁-L-NR₂ sequence canbe transcribed as a single chain (ssRNA). In alternative embodiments,the RNA nanostructure is comprised of two or more motifs, wherein thefirst nano-robot comprises a first motif, and the second nano-robotcomprises a second motif. In some embodiments, the first and secondmotifs is separately transcribed as two separate RNA chains. Portions ofthe two separate RNA chains can be hybridized to each other to form alinker, L. In some embodiments, the hybridization is a directhybridization between a portion of the first RNA chain which iscomplementary to a portion of the second RNA chain. In some embodiments,the hybridization is an indirect hybridization via a bridgeoligonucleotide wherein a portion of the sequence of one terminus of thebridge oligonucleotide is complementary to a portion of the sequence ofthe first RNA chain, and a sequence of the other terminus of the bridgeoligonucleotide is complementary to a portion of the sequence of thesecond RNA chain, wherein hybridization occurs between portions of eachof the first and second RNA chains and the bridging oligonucleotide. Incertain embodiments, the linker is pH-sensitive. The RNA duplex linkedto double nanobots can be responsive to an acidic environment fordehybridization. RNA duplex stability is pH sensitive as a lower pH isreduces the melting temperature of short RNA-duplexes.

In certain embodiments, the present invention provides for a RNAnanostructure as described herein where the first scaffoldself-assembles into a rectangular shape.

In certain embodiments, the present invention provides for a RNAnanostructure robot that further comprises a moiety R₄, wherein R₄ is acargo molecule. In some embodiments, R₄ is a cargo molecule and m is aninteger from 1 to 20.

The cargo molecule can be an aptamer, protein, or drug molecule. In someembodiments, the protein is an antigen. In some embodiments, the cargomolecule is operably linked to NR₂.

In some embodiments, R₄ is a fastener strand of DNA. As used herein, theterm “fastener strand” is a polynucleic acid which can enforce the firstor second RNA nanostructure scaffold into a 3-D shape (“origamistructure”). In some embodiments, R₄ is a pair of fastener strands whichcomprise DNA, and the pair of fasteners strands are capable of fasteningthe first or second scaffold into an origami structure, and n is aninteger from 1 to 20. In some embodiments, the fastener strand cancomprise an aptamer which binds to a locking protein. In some aspects,the locking protein is selected from: interferon-gamma (IFN-7), VEGF,and IDO. For example, in some embodiments, VEGF aptamers are attached tothe RNA nanocage, in which anti-VEGF-Receptor antibodies are encaged.Similarly, if an aptamer that binds IDO were attached to the structure,IDO inhibitors are encaged within the RNA-cage. In some embodiments, thefastener strands comprises a first strand and a second strand, whereinthe second strand of DNA comprises a sequence which is partiallycomplementary to the sequence of the first strand. Each of the fastenerstrands of DNA can comprise a first and a second strand of DNA. In someembodiments, a portion of one terminus of the first strand of fastenerDNA is complementary to a portion of one terminus of a second strand ofDNA. A portion of the other terminus of the first strand of fastener DNAis complementary to a portion of the ssRNA of NR₁ or NR₂. In someembodiments, a portion of one terminus of the second strand of fastenerDNA is complementary to a different portion of the ssRNA of NR₁ or NR₂.The fastener strands hybridize with their respective complements therebyconfiguring the RNA nanostructure into a 3-dimensional shape asdescribed herein (“RNA origami”). The first and second strand of DNA canbe selected from a sequence pair of the following oligonucleotides:

5′-FITC-labeled F50 and 3′-BHQ1-labeled Comp15;FITC-F50-48 and Comp15-48-Q; FITC-F50-73 and Comp15-73-Q;FITC-F50-97 and Comp15-97-Q; FITC-F50-120 and Comp15-120-Q;FITC-F50-144 and, Comp15-144-Q; or FITC-F50-169 and Comp15-169-Q;wherein the aforementioned oligonucleotideshave the following sequences: 5′-FITC-labeled F50: (SEQ ID NO: 10)5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGT GAATTGCG-3′;3′-BHQ1-labeled Comp15: (SEQ ID NO: 11)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; FITC-F50-48: (SEQ ID NO: 12)5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGT GAATTGCG-3′;Comp15-48-Q: (SEQ ID NO: 13)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-73(SEQ ID NO: 14) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGGAAATCAAAA-3′; Comp15-73-Q: (SEQ ID NO: 15)5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-97:(SEQ ID NO: 16) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGAACAAACTAT-3′; Comp15-97-Q: (SEQ ID NO: 17)5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-120:(SEQ ID NO: 18) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTACAAACAGTT-3′; Comp15-120-Q: (SEQ ID NO: 19)5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-144:(SEQ ID NO: 20) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTTCCTATTATT-3;; Comp15-144-Q: (SEQ ID NO: 21)5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-BHQ1′-3′; FITC-F50-169:(SEQ ID NO: 22) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTCACCTGAAA-3′; Comp15-169-Q: (SEQ ID NO: 23)5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′.

In some embodiments, this disclosure provides for a RNA nanostructurerobot wherein the first and second strands of fastener DNA are selectedfrom a sequence pair of the following oligonucleotides:

F50 and Comp15; F50-48 and Comp15-48; F50-73 and Comp15-73;F50-97 and Comp15-97; F50-120 and Comp15-120;F50-144 and, Comp15-144; or F50-169 and Comp15-169;wherein the aforementioned oligonucleotideshave the following sequences: F50: (SEQ ID NO: 24)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGA ATTGCG-3′; Comp15:(SEQ ID NO: 25) 5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; F50-48:(SEQ ID NO: 26) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGAATTGCG-3′; Comp15-48: (SEQ ID NO: 27)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; F50-73 (SEQ ID NO: 28)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGGAAA TCAAAA-3′; Comp15-73:(SEQ ID NO: 29) 5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-3′; F50-97:(SEQ ID NO: 30) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGAACAAACTAT-3′; Comp15-97: (SEQ ID NO: 31)5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-3′; F50-120: (SEQ ID NO: 32)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTACAA ACAGTT-3′; Comp15-120:(SEQ ID NO: 33) 5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-3′; F50-144:(SEQ ID NO: 34) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTTCCTATTATT-3′;; Comp15-144: (SEQ ID NO: 35)5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-3′; F50-169: (SEQ ID NO: 36)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTCAC CTGAAA-3′; Comp15-169:(SEQ ID NO: 37) 5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-3′.

In some embodiments, one or more of R3 and/or R4 is an aptamer thatspecifically binds a target molecule and comprises domain whichcomprises a sequence which is partially complementary to the sequence ofthe second strand, and m is an integer from 1 to 20.

In some embodiments, one or more of R₃ and/or R₄ is an RNA targetingstrand, wherein each targeting strand is operably linked to a targetingmoiety and to NR₁ or NR₂. As used herein, the term “targeting strand” isa polynucleic acid which can comprise an aptamer sequence. Thepolynucleic acid can further comprise DNA, RNA, modified DNA, modifiedRNA, or combinations thereof. The targeting moiety can be an aptamerthat specifically binds a target molecule. In some embodiments, theaptamer is specific for nucleolin. In some embodiments, the aptamer thatis specific for nucleolin is an F50 AS1411 aptamer having the sequence:

(SEQ ID NO: 38) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′.

In some embodiments, the aptamer can comprise any of the followingsequences:

T-1 (SEQ ID NO: 49) GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTCGATGGCCCACTACGTAAACCGTC; T-2 (SEQ ID NO: 50)GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTCGGTTTGCGTATTGGGAACG CGCG; T-11(SEQ ID NO: 51) GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTGACAGCATCGGAACGAACCC TCAG;T-12 (SEQ ID NO: 52) GGTGGTGGTGGTTGTGGTGGTGGTGGATTTTACTTTCAACAGTTTCTGGGATTT; T-205 (SEQ ID NO: 53)GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTACCAGTAATAAAAGGGATTC ACCA; T-206(SEQ ID NO: 54) GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTAATCAATATCTGGTCACAAA TATC;T-215 (SEQ ID NO: 55) GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTATAAATCCTCATTAAATGATATTC; or T-216 (SEQ ID NO: 56)GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTTATAAGTATAGCCCGGCCGT CGAG.

In some embodiments, the aptamer is any of the aptamers listed in Tables1 or 2.

TABLE 1 Aptamer sequences of the present invention. Aptamer Name TargetSequence (5′ to 3′) Macugen vascular CGGAAUCAGUGAAUGCUUAUACAUCCG (SEQ(Pegaptanib endothelial ID NO: 57) Sodium) growth As modified: factor(2′-deoxy-2′-fluoro)C-Gm-Gm-A-A-(2′-deoxy-2′-fluoro)U- (VEGF)(2′-deoxy-2′-fluoro)C-Am-Gm-(2′-deoxy-2′-fluoro)U-Gm-Am-Am-(2′-deoxy-2′-fluoro)U-Gm-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)U-Am-(2′-deoxy-2′-fluoro)U-Am-(2′-deoxy-2′-fluoro)C-Am-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)C-Gm-(3′→3′)-dT),5′-ester with α,α′-[4,12-dioxo-6-[[[5-(phosphoonoxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[ω-methoxypoly(oxy-1,2-ethanediyl)] (SEQ ID NO: 132) CL4 EGFRGCCUUAGUAACGUGCUUUGAUGUCGAUUCGAC AGGAGGC (SEQ ID NO: 58) E07 EGFRUGCCGCUAUAAUGCACGGAUUUAAUCGCCGUA GAAAAGCAUGUCAAAGCCG (SEQ ID NO: 59) 3.1 hTNFα CCCCGGGUUCUGUAUGAUCCGACCGGUCAGAU AAGACCAC (SEQ ID NO: 60) 7.5 hTNFα CGCAUCGUUUGCGUGGCGUGUCCGGGCGCCGA UUCGUAAA (SEQ ID NO: 61) 12hTNFα CUAGGCGGAUUGUUUCGAUUCUUUGCCUUGUC CCUAGUGC (SEQ ID NO: 62) 14.8hTNFα CGUAUAUACGGAUUAGGUUGUAGCUCAGACCA GUAAUGUC (SEQ ID NO: 63) 16.3hTNFα CGUGCUAGAUGCUACGAGUGGUCUCCUCACGU AGAAGGGG (SEQ ID NO: 64) 18.10hTNFα GGUCCCACAUAGGUUGGUCUUGUUGUAUGGGC UGUUUGCA (SEQ ID NO: 65)  1 hTNFαGUGUUUUGGGAGAGAAAAGGGGGAGCCUUUA CUUUGUUGG (SEQ ID NO: 66)  2 hTNFαGACGAUGUUAUCAGGGAGUUGGGAUCAUAUA GUCUUACAU (SEQ ID NO: 67)  4 hTNFαCGCAAGAGCCGCCCUAAUGGUUCAAUGGUAAC UGUAUAUG (SEQ ID NO: 68)  6 hTNFαGACUUCUUGUGCCAUUAUGAAUUAUUGCUAA UCCUCUUGA (SEQ ID NO: 69)  8.6 hTNFαAGGACGUACUUGGAAAAGAGGCGCGAAGAACC UGGUAUGU (SEQ ID NO: 70)  9 hTNFαUAGGACGUACUUGGAAAAGAGGCGCGAAGAA CCUGGUAUG (SEQ ID NO: 71) 10 hTNFαUGGCCACCUUGCCACUCUUCCUUGCAUAUUUU ACUCCCGC (SEQ ID NO: 72) 11.7 hTNFαCAAGCCGAGGGGGAGUAUCUGAUGACAAUUCG GAGCUCCA (SEQ ID NO: 73) 13.2 hTNFαUCAUGGUGUGUGAGUUAGCUCACGUGCCGUUU CGAAGGCG (SEQ ID NO: 74) 17.9 hTNFαCAUGGGCUAGACCGGCAUAAAACUGCUGUAGU UGCACGCC (SEQ ID NO: 75) 19 hTNFαUGGCCACCUUGCCACUCUUCCUUGCAUAUUUU ACUCCCGC (SEQ ID NO: 76) 20.4 hTNFαCGUUGUAGUAGUGGCUUGGGCAUAACUCAGU UAAACACUA (SEQ ID NO: 77)  5 hTNFαGACCGCGGAAAAGGAAGGAAUUAGAUACAAC GGAGAAGUG (SEQ ID NO: 78) 15 hTNFαGACCGCGGAAAAGGAAGGAAUUAGAUACAAC GGAGAAGUG (SEQ ID NO: 79) T1.7.5 hTNFαCGCAUCGUUUGCGUGGCGUGUCCGGGCGCCGA UUCGUAAA (SEQ ID NO: 80) T2.13.2 hTNFαUCAUGGUGUGUGAGUUAGCUCACGUGCCGUUU CGAAGGCG (SEQ ID NO: 81) T3.11.7 hTNFαCAAGCCGAGGGGGAGUAUCUGAUGACAAUUCG GAGCUCCA (SEQ ID NO: 82) T4.14.8 hTNFαCGUAUAUACGGAUUAGGUUGUAGCUCAGACCA GUAAUGUC (SEQ ID NO: 83) A38 VacciniaTACGACTCACTATAGGGATCCTGTATATATTTT virus GCAACTAATTGAATTCCCTTTAGTGAGGGTT(SEQ ID NO: 84) Alicaforsen ICAM-1 GCCCAAGCTGGCATCCGTCA (SEQ ID NO: 85)(ISIS 2302) AX102 PDGF-B 5′-dC-dC-dC-dA-dG-dG-dC-T-dA-dC-mG-HEG-dC-dG-(RNA) T-dA-mG-dA-mG-dC-dA-mU-mC-mA-HEG-T-dG-dA-T-mC-dC-T-mG-mG-mG-3′dT (SEQ ID NOS 86, 130, and 131, respectively)SL (2)-B VEGF CAATTGGGCCCGTCCGTATGGTGGGT (SEQ ID (DNA) NO: 87) RNV66VEGF TGTGLGGGTGGACGGGCCGGLTALA (L = LNA (DNA) guanine) (SEQ ID NO: 88)AS1411 Nucleolin GGTGGTGGTGGTTGTGGTGGTGGTGG (SEQ ID NO: 89) FCL-IINucleolin ggt ggTL ggt ggTD tgt ggt ggt ggdI gg (SEQ ID NO: 90)6L/12D/24_(dI) (2′-deoxyinosine (2′-dI) and D-/L-isothymidine(D-/L-isoT)) PNDA-3 Periostin ACGAGYYGYCGCAYGYGCGGYYCAGYCYGGYC (DNA)CYYCAGCACCGYACAACAA (SEQ ID NO: 91) NAS-24 VimentinCTC CTC TGA CTG TAA CCA CGC CTG GGA CAG (DNA)CCA CAC AGA AGT GTA GAC CTC GCG GAA TCGGCA TAG GTA GTC CAG AAG CC (SEQ ID NO: 92) YJ-1 (RNA) CEAGCGGAAGCGUGCUGGGCUAGAAUAAUAAUAA GAAAACCAGUACUUUCGU (SEQ ID NO: 93)AGE-apt AGE CGAAACCAGACCACCCCACCAAGGCCACTCGG (DNA)TCGAACCGCCAACACTCACCCCA (SEQ ID NO: 94) A-P50 NF-κBGATCTTGAAACTGTTTTAAGGTTGGCCGATC (RNA) (SEQ ID NO: 95) OPN-R3 OPNCGGCCACAGAAUGAAAAACCUCAUCGAUGUUG (RNA) CAUAGUUG (SEQ ID NO: 96) cy-aptHGC-27 CGACCCGGCACAAACCCAGAACCATATACACG (DNA)ATCATTAGTCTCCTGGGCCG (SEQ ID NO: 97) A9g (RNA) PSMAGGGACCGAAAAAGACCTGACTTCTATACTAAGT CTACGTTCCC (SEQ ID NO: 98) ESTAE-selectin CGCTCGGATCGATAAGCTTCGATCCCACTCTCC (DNA)CGTTCACTTCTCCTCACGTCACGGATCCTCTAGA GCACTG (SEQ ID NO: 99) M12-23 4-1 BBGGGAGAGAGGAAGAGGGAUGGGCGACCGAAC (RNA) GUGCCCUUCAAAGCCGUUCACUAACCAGUGGCAUAACCCAGAGGUCGAUAGUACUGGAUCCCCC C (SEQ ID NO: 100) OX40-apt OX40CAGUCUGCAUCGUAGGAAUCGCCACCGUAUAC (RNA) UUUCCCAC (SEQ ID NO: 101)CD28-apt CD28 CAGAGACTTCCAAAATAAAAGACTC (SEQ ID NO: 102); (RNA)CTGAAAGTTGCAAAATAAAAAACTC (SEQ ID NO: 103);CACTTACCACAATAACAAACGGGTG (SEQ ID NO: 104);CCTGCACCACAGGGAGACGGGGGCC (SEQ ID NO: 105);GATTAGACCATAGGCTCCCAACCCC (SEQ ID NO: 106);TCTGAGGTGCTCCTGCTTTGGAACT (SEQ ID NO: 107); andCAAGACCGTTATGTCGTGTGTACTT (SEQ ID NO: 108) De160 CTLA-4CCGACGTGCCGCAACTTCAACCCTGCACAACCA (RNA) ATCCGCC (SEQ ID NO: 109)PSMA-4-1 PSMA/4-1 GGGAGGACGAUGCGGAUCAGCCAUGUUUACGU BB-apt BBCACUCCUUGUCAAUCCUCAUCGGCAGACGACU (RNA) CGCCCGA (SEQ ID NO: 110)MP7 (DNA) PD-1 GTACAGTTCCCGTCCCTGCACTACA (SEQ ID NO: 111);GTACAGTTCCCGTCCTGCACTACA (SEQ ID NO: 112) aptPD-L1 PD-L1ACGGGCCACATCAACTCATTGATAGACAATGCG (DNA) TCCACTGCCCGT (SEQ ID NO: 113)R5A1 IL10R CTGTAATTGGCGTATGTAACCCAGGCACCAAAC (RNA)ACCCCAG (SEQ ID NO: 114) CL-42 IL4Rα AAAAAGCAACAGGGTGCTCCATGCGCATCGAA(RNA) CCTGCGCG (SEQ ID NO: 115) CD44-EpC CD44/GGGATGGATCCAAGCTTACTGGCATCTGGATTT AM aptamer EpCAMGCGCGTGCCAGAATAAAGAGTATAACGTGTGA (RNA)ATGGGAAGCTTCGATAGGAATTCGG (SEQ ID NO: 116);GCGACTGGTTACCCGGTCGTAA (SEQ ID NO: 117) TIM3Apt TIM3GGGAGAGGACCAUGUAGUCACUAUGGUCUUG (RNA) GAGCUAGCGGCAGAGCGUCGCGGUCCCUCCCGGGAGAGGACCAUGUACUGGUAGUUCUCUGU GCGACUCCUACAGAGAGUCGCGGUCCCUCCCGGGAGAGGACCAUGUACUGGGUUGUAGGGGG GCUCCUUAGGCAGAGCGUCGCGGUCCCUCCCGGGAGAGGACCAUGUACCCCGCAAUGCGGCCC CAGACUUCAACAGAGCGUCGGGUCCCUCCCGGGAGAGGACCAUGUACCGCAUGUGCGCGAGA GGUAGCGACAGAGCGUCGCGGUCCCUCCC(SEQ ID NO: 118) AptCTLA-4 CTLA-4 TCCCTACGGCGCTAACGATGGTGAAAATGGGCC(DNA) TAGGGTGGACGGTGCCACCGTGCTACAAC (SEQ ID NO: 119)

TABLE 2 Aptamer names and targets of the present invention. Aptamer NameTarget ARC126 (RNA) PDGF-B NOX-A12 (RNA) CXCL12 E0727 (RNA) EGFR CL428(RNA) EGFR KDI130 (RNA) EGFR TuTu2231 (RNA) EGFR TTA140,41 (DNA)Tenascin-C GBI-1042 (DNA) Tenascin-C GL21.T (RNA) Axl AGC03 (DNA) HGC-27BC15 (DNA) hnRNP A1 CD16α/c-Met-apt (RNA) CD16α/c-Met VEGF-4-1BB apt(DNA) VEGF/4-1BB

In some embodiments, this invention provides for a RNA nanostructurerobot, wherein the targeting strand comprises a domain comprising apolynucleotide sequence for attaching to NR₁ or NR₂.

In some embodiments, this invention provides for a RNA nanostructurerobot, wherein the first or second scaffold is configured to have arectangular sheet having four corners and is shaped into a tube-shape.In some embodiments, the dimension of the rectangular sheet is about 90nm×about 60 nm×about 2 nm. In some embodiments, one or more targetingstrands are positioned at one or more corners of the rectangular sheet.In some embodiments, the tube-shaped origami structure has a diameter ofabout 19 nm.

In some embodiments, one or more of R₃ and/or R₄ is a capture strand. Asused herein, the term “capture strand” is a polynucleic acid which is inpart complementary to a homopolynucleic acid sequence. In someembodiments, the capture strand can bind to a poly(A) region in NR₁ orNR₂. In some embodiments, the capture strands is operably linked to atherapeutic agent. The capture strand can comprise a modified nucleosideor ribonucleoside which can bind to the therapeutic agent. In someembodiments, the capture strand comprises an RNA loop. In someembodiments, the capture strand comprises poly(U). In some embodiments,the capture stand comprises a sequence comprising an amino-modifiedribonucleoside.

In some embodiments, one or more of R₃ and/or R₄ is a therapeutic agentwhich is operably linked to the RNA nanostructure.

In some embodiments, the RNA nanostructure comprises one single-strandedRNA (ssRNA) molecule which forms at least one paranemic cohesioncrossover.

In some embodiments, the RNA nanostructure is immuno-stimulatory.

In some embodiments, the RNA nanostructure comprises a nucleic acidsequence having at least about 90% sequence identity to SEQ ID NO:1 orSEQ ID NO: 9. In some embodiments, the RNA nanostructure comprises anucleic acid sequence having at least about 95% sequence identity to SEQID NO:1 or SEQ ID NO: 9. In some embodiments, the RNA nanostructurecomprises SEQ ID NO:1 or SEQ ID NO: 9. In some embodiments, the RNAnanostructure consists of SEQ ID NO: 1 or SEQ ID NO: 9.

In some embodiments, one or more of R₃ and/or R₄ is a peptide thatcomprises a positively-charged moiety which comprises from about 10positively-charged amino acids. The the positively-charged moiety is apeptide comprising 10 lysine residues.

In some embodiments, one or more of R₃ and/or R₄ is a protein, whereinthe protein is selected from: tumor targeting peptide (TTP), a humancancer peptide, or calreticulin protein. In some embodiments, theprotein is calreticulin protein to RNA-origami to engage interactionsbetween tumor cells and macrophages or dendritic cells for enhancedantigen presentation and stimulation of antigen-specific T cells. Insome embodiments, the protein is Human cancer peptide NY-ESO-1 or Mucd.In some embodiments, the TTP is CTKD-K10 having the sequence:CTKDNNLLGRFELSGGGSKKKKKKKKKK (SEQ ID NO: 3).

In some embodiments, this invention provides for a pharmaceuticalcomposition comprising a RNA nanostructure robot described herein and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition further comprises at least one therapeuticagent.

In some embodiments, this invention provides for a method of treatingcancer in a subject, comprising administering to the subject atherapeutically effective amount of the composition described herein. Insome embodiments, the cancer is breast cancer, ovarian cancer, melanomaor lung cancer.

In some embodiments, this invention provides for a method of inhibitingtumor growth in a subject, comprising administering to the subject atherapeutically effective amount of a composition described herein.

In some embodiments, this invention provides for the use of the RNAnanostructure robot as described herein or a composition as describedherein for the manufacture of a medicament for inducing a tumor necrosisresponse in a subject.

In some embodiments, this invention provides for the use of the RNAnanostructure robot as described as described herein or a composition asdescribed herein for inducing a tumor necrosis response.

In some embodiments, this invention provides for the use of the RNAnanostructure robot as described as described herein or a composition asdescribed herein for the manufacture of a medicament for treating adisease or disorder in a subject.

In some embodiments, this invention provides for the use of the RNAnanostructure robot as described as described herein or a composition asdescribed herein for the prophylactic or therapeutic treatment a diseaseor disorder.

In certain embodiments, the present invention provides a complexcomprising an RNA nanostructure and at least one diagnostic and/ortherapeutic agent operably linked to the RNA nanostructure.

In certain embodiments, the RNA nanostructure comprises onesingle-stranded RNA (ssRNA) molecule, wherein the ssRNA molecule formsat least one paranemic cohesion crossover, and wherein the RNAnanostructure has immuno-stimulatory properties.

In certain embodiments, the RNA nanostructure comprises onesingle-stranded RNA (ssRNA) molecule, wherein the ssRNA moleculecomprises a plurality of regions of double helices, wherein at least twoof the plurality of regions of double helices form a paranemic cohesioncrossover, and wherein the RNA nanostructure has immuno-stimulatoryproperties.

In certain embodiments, the e ssRNA molecule comprises at least twoparallel double helices.

In certain embodiments, about 60-99% of the RNA nanostructure is doublestranded and about 1-40% of the RNA nanostructure is single stranded.

In certain embodiments, about 95% of the RNA nanostructure is doublestranded and about 5% of the RNA nanostructure is single stranded.

In certain embodiments, the RNA nanostructure comprises rectangularorigami nanostructure.

In certain embodiments, the RNA nanostructure comprises a nucleic acidsequence about 1500 to about 2500 nucleotides in length.

In certain embodiments, the RNA nanostructure comprises a nucleic acidsequence having at least about 75% sequence identity to SEQ ID NO:1 orSEQ ID NO: 9.

In certain embodiments, the RNA nanostructure comprises a nucleic acidsequence having at least about 85% sequence identity to SEQ ID NO:1 orSEQ ID NO: 9.

In certain embodiments, the RNA nanostructure comprises a nucleic acidsequence having at least about 95% sequence identity to SEQ ID NO:1 orSEQ ID NO: 9. In certain embodiments, the RNA nanostructure comprises anucleic acid sequence having at least about 90% sequence identity to SEQID NO:1 or SEQ ID NO: 9.

In certain embodiments, the RNA nanostructure comprises SEQ ID NO:1 orSEQ ID NO: 9.

In certain embodiments, the RNA nanostructure consists of SEQ ID NO:1 orSEQ ID NO: 9.

In certain embodiments, the nucleic acid sequence of the RNAnanostructure is about 1500 to about 2500 nucleotides in length.

In certain embodiments, the RNA nanostructure comprises at least oneparanemic cohesion crossover.

In certain embodiments, the diagnostic or therapeutic agent is a peptidecomprises a positively-charged moiety.

In certain embodiments, the positively-charged moiety is a peptidecomprising from about 5 to 20 positively-charged amino acids.

In certain embodiments, the positively-charged moiety is a peptidecomprising from about 8 to 12 positively-charged amino acids.

In certain embodiments, the positively-charged moiety is a peptidecomprising from about 10 positively-charged amino acids.

In certain embodiments, the positively-charged moiety is a peptidecomprising 10 lysine residues.

In certain embodiments, the protein is tumor targeting peptide (TTP), ahuman cancer peptide, an infectious agent peptide, or calreticulinprotein.

In certain embodiments, the infectious agent peptide is specificepitopes for CD8+ T cells involved in the immunity against influenza,HIV, HCV, and other infectious agents.

In certain embodiments, the protein is calreticulin protein.Calreticulin protein allows the RNA-origami to engage interactionsbetween tumor cells and macrophages or dendritic cells for enhancedantigen presentation and stimulation of antigen-specific T cells.

In certain embodiments, the protein is Human cancer peptide NY-ESO-1 orMuc.

In certain embodiments, the present invention provides a pharmaceuticalcomposition comprising the complex described herein and apharmaceutically acceptable carrier.

In certain embodiments, the present invention provides thepharmaceutical composition described herein and further comprising atleast one therapeutic agent.

In certain embodiments, the at least one therapeutic agent is achemotherapeutic drug.

In certain embodiments, the chemotherapeutic drug is doxorubicin.

In certain embodiments, the present invention provides a method ofinducing an immune response a subject (e.g., a mammal, such as a human),comprising administering to the subject an effective amount of a complexor a composition as described herein.

In certain embodiments, the present invention provides a method oftreating a disease or disorder in a subject, comprising administering tothe subject a therapeutically effective amount of a complex or acomposition as described herein.

In certain embodiments, the disease or disorder is cancer.

In certain embodiments, the cancer is breast cancer.

In certain embodiments, the cancer is colon cancer.

In certain embodiments, the method further comprises administering atleast one therapeutic agent to the subject.

In certain embodiments, the at least one therapeutic agent is a tumortargeting agent.

In certain embodiments, the tumor targeting agent is a monoclonal tumorspecific antibody or an aptamer.

In certain embodiments, the present invention provides a method ofenhancing/increasing pro-inflammatory cytokines in a subject (e.g., amammal, such as a human), comprising administering to the subject aneffective amount of a complex or a composition as described herein.

In certain embodiments, the present invention provides a method ofactivating immune cells by specific triggering of TLR3 signaling pathwayin a subject (e.g., a mammal, such as a human), comprising administeringto the subject an effective amount of a complex or a composition asdescribed herein.

In certain embodiments, the present invention provides a method ofslowing or suppressing tumor growth in a subject (e.g., a mammal, suchas a human) as compared to a control subject, comprising administeringto the subject an effective amount of a complex or a composition asdescribed herein.

In certain embodiments, the present invention provides a method toelevate levels of anti-tumor proinflammatory cytokines in a subject(e.g., a mammal, such as a human) with a tumor as compared to a controlsubject, comprising administering to the subject an effective amount ofa complex or a composition as described herein.

In certain embodiments, the present invention provides a method todecrease levels of anti-inflammatory cytokines in a subject (e.g., amammal, such as a human) with a tumor as compared to a control subject,comprising administering to the subject an effective amount of a complexor a composition as described herein.

In certain embodiments, the present invention provides the use of acomplex or a composition as described herein for the manufacture of amedicament for inducing an immune response in a subject (e.g., a mammal,such as a human).

In certain embodiments, the present invention provides a complex or acomposition as described herein for inducing an immune response.

In certain embodiments, the present invention provides a use of acomplex or a composition as described herein for the manufacture of amedicament for treating a disease or disorder in a subject.

In certain embodiments, the present invention provides a complex or acomposition as described herein for the prophylactic or therapeutictreatment of a disease or disorder.

In certain embodiments, the present invention provides a kit comprisinga complex or a composition as described herein and instructions foradministering the RNA anostructure/composition to a subject to induce animmune response or to treat a disease or disorder. In certainembodiments, the kit further comprises at least one therapeutic agent.The kits of the invention can include any combination of thecompositions and/or vaccines disclosed above and suitable instructions(e.g., written, audiovisual). In one embodiment, the kit includes apharmaceutical composition or vaccine that is packaged along withinstructions for use and any instrument useful in administering thecompositions. For example, the kits of the invention can include one ormore of: diluents, gloves, vials or other containers, pipettes, needles,syringes, tubing, sterile cloths or drapes, positive and/or negativecontrols, and the like.

As shown in FIG. 20, in some embodiments the RNA nanostructure polymericrobot 1 comprises a first nano-robot (NR₁) 8, a second nano-robot (NR₂)9, a linker (L) 3, a moiety (R₃) 2 and n is 1 or more, and the moiety R₃is in contact with NR₁. In some embodiments, the moiety R₃ is an antigenpeptide. A second optionally heterogeneous series of moiety R₄ (4, 5, 6,7) comprise a nucleolin-binding or other cancer tumor marker aptamers 5wherein m is 2 or more, an aptamer-target bound lock 6, optionally acargo molecule 7, and optionally a checkpoint antagonist 4. In someembodiments, the linker (L) 3 comprises of a double-strandedoligonucleotide helix. In some embodiments, the antigen peptides aretumor antigen peptides. In some embodiments, the tumor antigen peptidesare TTP. In some embodiments, the cargo molecule 7 is a therapeuticagent. In some embodiments, the cargo molecule 7 is an antibody. Theantibody can be an anti-PD-1 antibody. The antibody can further comprisea linkage to a polylysine chain. In some embodiments, the checkpointantagonist is an anti-PD-1 aptamer. In some embodiments, the checkpointantagonist is an anti-PD-1 antibody. In some embodiments, the firstnano-robot (NR₁) 8 is an immune-adjuvant RNA robot exhibitingimmune-stimulatory properties. In some embodiments, the aptamer-targetbound lock 6 is an INF-γ aptamer bound to INF-γ. In some embodiments,the second nano-robot (NR₂) 9 is configured to be in a cylindricalconformation which is operably openable via the aptamer-target boundlock 6. The RNA nanostructure polymeric robot 1 induces activation of Tcells 11 at antigen-presenting cells 9 by contacting the firstnano-robot (NR₁) 8 comprising a moiety R₃ wherein the moiety is anantigen peptide 2, with a T cell 11. In some embodiments, the secondnano-robot (NR₂) 9 interacts with the tumor environment 13. In someembodiments, the aptamer-target bound lock 6 is opened when the targetis a compound, biomarkers, or protein present in high regionalconcentration in the tumor environment 13. The aptamer-target bound lock6 competitively binds with the free target in the tumor environment 13,resulting in the unlocking of the second nano-robot (NR₂) 9. The openingof the second nano-robot (NR₂) 9 exposes the optional cargo molecule 7,and the optional checkpoint antagonist 4, to the tumor environment. Whenthe cargo molecule 7 is a therapeutic agent and/or the checkpointantagonist 4 is present, the therapeutic agent and/or checkpointantagonist effect tumor necrosis in the tumor environment 13.

As shown in FIG. 21, in certain embodiments the scaffold is an unfoldedrectangular RNA origami structure 20 having fasteners 50 extending fromthe edges that is joined, aptamer-containing targeting strands and atherapeutic agent-RNA conjugate capture strand. In certain embodiments,the RNA origami structure 20 also has aptamer-containing targetingstrands 30 attached thereto.

As shown in FIG. 22 in certain embodiments the aptamer-containingtargeting strand 30 containing an aptamer portion 31 and an attachingRNA strand portion 32.

As shown in FIG. 23 in certain embodiments the fastener 50 has two arms51 and 52, and having a quencher moiety 54 attached to one arm 52 of theY-structure and a fluorophore moiety 55 attached to the second arm 51 ofthe Y-structure by means of a linker 53.

As shown in FIG. 24 in certain embodiments the therapeutic agent-RNAconjugate capture strand 15 has a ssRNA attachment strand 10 and atherapeutic agent payload 11. As shown in FIG. 25 in certain embodimentsthe therapeutic agent payload 11 is operably linked to an imaging agent12. As shown in FIG. 26 in certain embodiments the ssRNA attachmentstrand 10 is linked to a therapeutic agent payload 11 by means of alinker 14. As shown in FIG. 27 in certain embodiments therapeuticagent-RNA conjugate capture strand having a ssRNA attachment strand thatis linked to a therapeutic agent payload by means of a linker, where thetherapeutic agent payload is operably linked to an imaging agent 12.

As shown in FIGS. 28-31, in certain embodiments the unfolded rectangularRNA origami structure has one to four therapeutic agent-RNA conjugatesoperably linked to the origami structure. The therapeutic agent-RNAconjugates can be attached to either the “top” or the “bottom” (or both)of the origami structure, such that when the origami structure is rolledinto a tube, the therapeutic agent-RNA conjugates can be designed to beeither on the inside or outside of the tube.

FIG. 32 depicts an exploded view of the RNA origami structure 20,detailing the hybridization of a single stranded RNA scaffold strand 60and staple strands 70, and the interaction of the two staple strands.

As shown in FIG. 33 in certain embodiments, the tube-shaped RNA origamistructure have therapeutic agent-RNA conjugates 10 positioned on theoutside of the tube-shaped RNA origami structure.

As shown in FIG. 34 in certain embodiments, the tube-shaped RNA origamistructure having therapeutic agent-RNA conjugates 10 positioned on theinside of the tube-shaped RNA origami structure.

As shown in FIG. 35, a tube-shaped RNA origami structure 20 havingtherapeutic agent-RNA conjugates 15 is positioned on the inside of thetube-shaped RNA origami structure, having aptamer-containing targetingstrands 30 positioned at the ends of the tube, and illustrating thefasteners 50 joining the edges of the RNA origami structure so as toform a tube shape.

As shown in FIG. 36, a first nano-robot 8 further comprises asingle-stranded RNA sequence which forms part of the linker 3 a. Asecond nano-robot 9 further comprises a single-stranded RNA sequencewhich forms part of the linker 3 b. The sequence of linker 3 a iscomplementary, in whole or in part, with the sequence of linker 3 b suchthat hybridization forms linking the first nano-robot 8 to the secondnano-robot 9. In some embodiments, the sequence of linker 3 a is notcomplementary to the sequence of linker 3 b. In some embodiments, thelinker further comprises a bridging oligonucleotide 3 c, wherein oneterminus of the the sequence of the bridging oligonucleotide 3 c iscomplementary to the sequence of linker 3 a, and the other terminus ofthe sequence of the bridging oligonucleotide 3 c is complementary to thesequence of linker 3 b. The oligonucleotides can comprise DNA, RNA, orcombinations thereof. In some embodiments, the oligonucleotides arepH-sensitive such that hybridization and/or the oligonucleotide sequencedenatures at the low pH of the tumor environment. In some embodiments,the sequence of the briding oligonucleotide 3 c is complementary to theDNA sequence within the tumor. In some embodiments, the sequence of thebriding oligonucleotide 3 c is complementary to the RNA sequenceoverexpressed within the tumor environment.

EXAMPLES

The invention will now be illustrated by the following non-limitingExamples.

Example 1

As described in FIG. 1, a plasmid containing an ssRNA origami gene waslinearized and the ssRNA was in vitro transcribed using T7 RNApolymerase. The purified RNA molecule was then self-assembled into thessRNA origami nanostructure. The properly-folded RNA origami was shownto be resistant to nuclease digestion and contained regions of bothdsRNA and ssRNA, which may serve as pathogen associated molecularpatterns. Specifically, in vitro RNase digestion experiments wereconducted and the RNA origami was found to exhibit higher nucleaseresistance than the unfolded ssRNA with the same sequence as the RNAorigami (FIG. 2). In addition, the immuno-stimulating effects of RNAorigami was tested using an ex vivo splenocyte stimulation assay andenhanced stimulatory activity mediated by RNA origami over PolyIC wasobserved (FIG. 3-FIG. 5). Similar to the in vitro findings onstimulation, an intraveneous injection of RNA origami through aretro-orbital route resulted in a transient elevation of IFNa/b in mice(FIG. 6).Upon prolonged incubation, the RNA origami were also found toreduce the viability of some tumor cells in vitro (FIG. 7). As shown inFIG. 8, the RNA origami acted as a TLR3 agonist in the HEK-Blue™-mTLR3reporter cell line. Finally, anti-tumor immunity was evaluated in vivousing an A20-iRFP model, which allowed tumor growth to be tracked invivo (FIG. 9-FIG. 10). In these experiments, mice were eitheradministered an antibody alone or the antibody in combination with RNAorigami. As shown in FIG. 9 and FIG. 10, tumor reduction was observedupon treatment with RNA origami, which was greater than with theadministration of antibody alone.

Taken together, these results indicate that the RNA origami can functionas agonists of pattern recognition receptors, such as TLR3 and TLR7 inimmune cells, and serve as a new line of adjuvants. By using anestablished mouse tumor model, this platform may be further explored forthe construction of tumor-specific vaccines.

Materials and Methods

Materials

Restriction endonucleases EcoRI (5,000 units), XhoI (5,000 units) andHindIII (5,000 units), T7 and T3 RNA polymerases (5,000 units), NEB10-beta competent E. coli were purchased from NEW ENGLAND BIO LABS INC.Pureyield plasmid miniprep system and the Wizard SV Gel and PCR Clean-UPSystem were purchased from Promega (www.promegA.com). RNA Clean andConcentrator-25 was purchased from Zymo Research (www.zymoresearch.com).

RNA Nanostructure Design

RNA rectangle origami nanostructure and RNA sequence were designed usingthe Tiamat software (Yanlab.asu.edu/Tiamat.exe).

Artificial RNA sequence was generated by using the following criteria inthe Tiamat software: (1) Unique sequence limit: 8 nt; (2) Repetitionlimit: 6-8 nt; (3) G repetition limit: 4 nt; (4) GC content: 0.45-0.55.Once sequences were generated, a few nucleotides were adjusted toeliminate the restriction enzyme targeting sequences (e.g. by EcoRI,EcoRV, HindII and XbaI) for cloning purposes. A T7 promoter sequencefollowed with three consecutive Gs were manually incorporated onto the5′ end of the DNA template (SEQ ID NO: 2) in order to facilitateefficient in vitro transcription reaction. The dsDNA template wassynthesized by BioBasic Inc. and cloned into the pUC19 vector throughEcoRI and HindII restriction sites.

RNA Strand Synthesis

The plasmid containing the ssRNA nanostructure gene was linearized byusing a HindII enzyme (New England Biolabs) and the linear plasmid waspurified by using a Phenol/chloroform extraction and ethanolprecipitation. The in vitro transcription reaction was carried out byusing the T7 RiboMAX Express Large Scale RNA Production System(Promega), following the manufacturer's instructions. For inosinecontaining RNA preparation, additional 5 mM Inosine-5′-triphosphate(TriLink BioTechnologies) was added to the in vitro transcriptionreaction. The RNA molecules were then purified via a RNA Clean &Concentrator-25 kit (Zymo Research). After purification, the ssRNA wasannealed using the same program as ssDNA origami. The RNA moleculeproduced from DNA template SEQ ID NO: 2 had the RNA sequence of SEQ IDNO: 1.

RNA Origami Nanostructure Assembly

The purified RNA molecule was diluted to 20 nM in 1×PBS buffer (20 mMSodium phosphate, 130 mM Sodium chloride, pH 7.4). The resultingsolution was annealed from 65° C. to 25° C. with a cooling ramp of 1° C.per 20 minutes to form the desired structures. The assemble RNA origaminanostructure was concentrated to the desired concentration using anAmicon Ultra-0.5 mL centrifugal filter (Millipore, 100 kDa cut off).

Atomic Force Microscope characterization

RNA origami was imaged in “ScanAsyst mode in fluid,” using a DimensionFastScan microscope with PEAKFORCE-HiRs-F-A tips (Bruker Corporation).After annealing, 2 μl of each sample was deposited onto a freshlycleaved mica surface (Ted Pella, Inc.), and left to adsorb for 1 minute.Then, 80 μl of 1×TAE-Mg buffer and 2 μl 100 mM of a NiCl2 solution wasadded onto the mica, and 40 μl of the same buffer was deposited onto themicroscope tip. The samples were then scanned by following themanufacturer's instructions.

Animals

Female BALB/c mice were obtained from Charles River Laboratories andmaintained in a pathogen-free animal facility at the Arizona StateUniversity Animal Resource Center. All mice were handled in accordancewith the Animal Welfare Act and Arizona State University InstitutionalAnimal Care and Use Committee (IACUC). Before experimental treatment,the mice were randomly distributed in cages and allowed to acclimate forat least 1 week prior to vaccination.

Splenocyte Isolation and Stimulation

Mice were euthanized with carbon dioxide asphyxia, and the spleens wereremoved and sterilized by quickly dipping in 70% ethanol for 1 s beforetransfer to sterile RPMI-1640 medium supplemented with 10% fetal bovineserum (FBS) in the biosafety cabinet. Spleen was cut on one end, and athin, sealed L-shaped glass tube was used to push spleen marrows out.The extracted spleen cells were pelleted and washed by spinning at 380×gfor 3 min in the sterile RPMI-1640 medium described above, and red bloodcells were depleted by ACT lysis buffer (combination of 0.16M NH4Cl and0.17 M Tris [pH 7.65] at a volume ratio of 9:1, pH adjusted to 7.2 with1 M HCl, and filter sterilized). After washing twice in RPMI-1640 mediumsupplemented with 10% FBS and antibiotics, the splenocytes were seededin 12-well plates at a density of 4×10⁶ cells/mL. RNA origami,Inosine-incorporated RNA origami, or other adjuvants are added into eachwell at desired concentrations (5 μg/mL, 0.5 μg/mL, or 0.05 μg/mL), 50ng/mL lipopolysaccharide (LPS) was added to the positive control welland Polymyxin B (PMB) is added into each well except for the LPS alonewell at final concentration of 100 μg/mL to prevent endotoxincontamination. 24 hours or 48 hours after stimulation, cells wereharvested, labelled for surface markers, and analysed by flow cytometry.

Flow Cytometry

Stimulated splenocytes were harvested by spinning down at 380×g for 3min, and supernatants were saved for cytokine analysis. Pelleted cellswere washed once with 1×PBS, and labeled with Zombie Violet viabilitydye (Biolegend) at room temperature for 15 min. After washing twice instaining buffer (1×PBS, 2% BSA, 0.01% sodium azide), cells wereincubated in the following antibody cocktail containing FcR block: (a)FITC anti-mouse CD4, PE anti-mouse CD3, PE/Cy5 anti-mouse CD69, andPE/Cy7 anti-mouse PD1; b) FITC anti-mouse CD11b, PE anti-mouse CD86,PE/Cy5 anti-mouse B220, and PE/Cy7 anti-mouse CD11c. After 30 minincubation at 4° C., cells were washed twice in staining buffer andresuspended in 200 uL staining buffer. Then each sample was analyzed ona FACSAria II instrument at Biodesign Institute, Arizona StateUniversity. Live cells were defined as Zombie Violet-low cellpopulation, and CD4 T cells were gated as CD3+CD4+ live cells, CD8 Tcells were gated as CD3+CD4-live cells. Percentage of CD69+ cells in CD4T cell population and CD8 T cell population were plotted for T cellstimulation measurement Plasmacytoid dendritic cells (pDC) were definedas CD11b−CD11c+B220+ live cells, and conventional dendritic cells (DC)were defined as CD11b+CD11c+ cells. Mean fluorescent intensity of CD86in each DC cell population is plotted as an indicator of DC stimulationstatus.

Cytokine Analysis

Cytokine release in ex vivo splenocyte cell culture supernatant wasmeasured by the mouse Procarta IFN 2-plex featured assay of EveTechnologies (catalog no. MIFN-02-103). For serum cytokine analysis, 100uL of RNA origami (25 μg), PolyIC (25 μg) or 1×PBS were i.v. injected tonaive mice through retro-orbital route, and mouse serum were collectedat 3 hr, 6 hr, and 24 hr post injection by cheek-vein bleeding. Bloodwas spin down at 7000 rpm for 10 min at 4° C., and measured by the mouseProcarta IFN 2-plex featured assay of Eve Technologies (catalog no.MIFN-02-103).

Cell Viability Test

Viability of cells after incubation with RNA origami was analyzed by MTTassay, (Vybrant®MTT cell proliferation assay kit from Thermo Fisher)following manufacture's protocol. Camptothecin (Sigma-Aldrich, catalogno. C9911) at final concentration of 5 μM served as the positivecontrol, because it induces apoptosis.

TLR3 Agonist Test

A reporter cell line expressing mouse TLR3, HEK-Blue™ mTLR3 cells, waspurchased from Invivogen. Agonist activity of RNA origami and otheradjuvants were quantified by the absorbance of HEK-Blue medium afterco-incubation of these adjuvants with cells, following manufacture'sprotocol. ssRNA40/LyoVec™ purchased from Invivogen served as negativecontrol.

A20-iFRP-OVA Tumor Model

A20, mouse B cell lymphoma cells, were transduced with lentiviral vectorthat was constructed to express near-infrared fluorescent protein (FIG.11), iRFP, and oval albumin using LENTI-Smart™ transduction kit fromInvivogen by following manufacture's protocols. Cell-sorting was carriedout on BD FACSAria II at Biodesign Institute, Arizona State University,to isolate A20-iRFP cells with the top 1% fluorescent intensity forsubsequent cell culture. Bright and stable expression of iRFP in A20cells were confirmed by flow cytometry and Pearl small animal imagingsystem (LI-COR, San Diego, Calif.). For tumor inoculation, BALB/c micewere shaved at the left flank and injected s.c. with 10×10⁶ A20-iRFPcells. 7-10 days post injection, mice were imaged under the Pearl smallanimal imaging system, and mice bearing tumors of similar near-infraredintensities were randomized into different groups for subsequenttreatments.

For treatment, mice were injected with 25 ug RNA origami in 50 uL PBS,or 50 uL PBS through intratumor injection on day 0. Anti-PD1 antibody(Biolegend, catalog no. 114108) were injected into mouse tumors on day 2and day 4, at a dose of 2.5 ug per injection. Tumor growth were trackedevery other day and tumor size was quantified by measuring thenear-infrared fluorescent intensity using Image Studio™ software fromLI-COR. The results showed that at day 10 post treatment, the cohorttreated with RNA origami with the anti-PD1 antibody exhibited a smallertumor size than the cohort treated with the anti-PD1 antibody alone(FIG. 10). In addition, the survival rate was surprisingly greater forthe cohort treated with RNA origami with the anti-PD1 antibody (rounddots) compared to the cohort treated with the anti-PD1 antibody alone(square dots) (FIG. 10). The cohort treated with the combination of RNAorigami with the anti-PD1 antibody only had a 5% drop in survival,whereas the cohort treated with the anti-PD1 antibody alone had a 50%drop in survival at 25 days post-treatment.

RNA Nanostructure Sequence for One Embodiment of NR₁

(SEQ ID NO: 1) GGGAGAGAGCUCGAGCGAACACUAGCCACUUGAUCACGCUGAGCGCUCGUACAAUGAAACACAGGUGUGUCAGUGCUAUGCACGUUCGAAGAGCUGUAUCAGCGUUCGUGUGAAUGAGUUCAACGGAGUGUUGACUAAGCCGGUUGCUACAUUUCUGUAGCACACAUAGUCAAGAUUUGCACCAGACGAUACUCUCCCUCAGUCCUGUUUAUGCAAGUCGUCGUAGUCCUGACGUACUUCCUAAGCUCGUCACUGUACUGAUGAUUCCACUGAUCAAGAUGCACGUAUCUUCAGUUUCCUGAAGAUCGGAGUAGGCACUAUAAUCGACAAGUAACGCUUACGAUUCCAUCACGAGUGACUUACCUGAACCAUAACUGACAAGGGACCACGCAGAGGUCAUACUCACAGGACUUCAAAUCUUGAGUCGGGUUCGAUCAUUUCUGAUCGAGACACCAGUGUGAGGUAAUCGUACGUCACUUGAUAGGAGCUCUAAGUAGAGUUGAGAGCCUGUUAACUAGACACGAGUAACGAGGUUAGCCUGUACGAGAUAUCGGGCUAUAGUGCGGACACGAUUGCACCAUUUCUGGUGCAACGAAGGUGAGCAUGUAUGGACAGGUCAGUGUGACUCAAGUCGAUAGUCCAAGUAGGUUAUCGACUCGCAUAGCUCAAUGACUGUCAUCGCCAGAGUAUCUAGGUGUCUACCUCACGAAUCGCGUCGUUACAUUUCUGUAACGCUCAUACCGUGCUGAUCUAUGGGACACGUCGCUUAUUCUUGGGUCAUGACAGUUGCCACAAACAAGGCACGACCUCACACCUGCGAACUUCAAGCGUUAGGCUGACGUUACAUGCUUGCGUGCACUGAUUCGUUUCCGAAUCAGAGACCUACGAAGCCAGAGUUCGUUCACUAUCAUAAGUGCACUGAUGCAUUUGUGCCAACAUUGAAGGCAUCGAGAUAAACAGCCGUCUUAAUCAAGUGAGCACCUGAGAUCAGCAUGAUUCGUCUAUUUCUAGACGAAUCAACUUCCAUUCAGGUGCCUUGCUACUUAAGACGGGAUUAACUCUCGAUGCAACGUGCAUUGGCACAACUCGUGAUGUGCACUUUCACACUGGAACGAACUCUGGCUUCGUAGGUCUGUUUGUCAUUUCUGACAAACUGCACGCACUGUUAGUACGUCAGCCACUUAACCGAAGUUCGUCAUAAGUAGGUCGUGCGACUACGAUGGCAACUUCUACUUACCAAGAAUAAGCGACGUGUCCCAUAAUGGAAGUCGGUAUGAGGUAUGACUUUCGUCAUACACGCGAUUCCACAAUGUGACACCUAACGUUUGAGGCGAUGACCUGAUACAAGCUAUGCAUGGUUCAAACCUACUUGGACUAUCGACUUGAGAUGAUAGUACCUGUCCAACUAACAGCACCUUCGAUACCUCGUUUCCGAGGUAUUCGUGUCCUGUGUCAGGCCCGAUAUUAAUGUGUGGCUAACCCUUAGGAACGUGUCUAGUUAACAGGCUCUCAACGUCAUGACGAGCUCCUAGUAGCAAGCGUACGAUACAUUGUGACUGGUGUCUACUGGAUUUCUCCAGUAACCCGACUCCGACUACAAAGUCCUGACUCAUUCACCUCUGCGUGGUCCCUUGUCAGUUGAGUCGAUGGUAAGUCAAUGCAUCAGGAAUCGUGGUUAAGUCUUGUCGAUCUGACACACUACUCCGCUGUCCUGUUUCCAGGACAGACGUGCAUUAGCAGUUGUGGAAUCAUCAGUACAGUGACGAGUCGUUACUGUACGUCAGCUUGUUUGCGACUUGCAGUUAAUCGACUGAGGGUCAAACGUGUCUGGUGUGUAGUCGGACUAUGUGACGUUCAUUUCUGAACGUACCGGCUUAGUCAACACUCCGUUGAUGAGUAUGACACGAACGAGUCAUUGGCUCUUCGCUUCAAUGUAGCACUGAACUUAUGAUGUUUCAUACACAUUACGCUCAGCGAACUGCUAUGGCUAGUGUUCGGAU CC

DNA Sequence Encoding RNA Nanostructure Having SEQ ID NO: 1

(SEQ ID NO: 2) GGGAGAGAGCTCGAGCGAACACTAGCCACTTGATCACGCTGAGCGCTCGTACAATGAAACACAGGTGTGTCAGTGCTATGCACGTTCGAAGAGCTGTATCAGCGTTCGTGTGAATGAGTTCAACGGAGTGTTGACTAAGCCGGTTGCTACATTTCTGTAGCACACATAGTCAAGATTTGCACCAGACGATACTCTCCCTCAGTCCTGTTTATGCAAGTCGTCGTAGTCCTGACGTACTTCCTAAGCTCGTCACTGTACTGATGATTCCACTGATCAAGATGCACGTATCTTCAGTTTCCTGAAGATCGGAGTAGGCACTATAATCGACAAGTAACGCTTACGATTCCATCACGAGTGACTTACCTGAACCATAACTGACAAGGGACCACGCAGAGGTCATACTCACAGGACTTCAAATCTTGAGTCGGGTTCGATCATTTCTGATCGAGACACCAGTGTGAGGTAATCGTACGTCACTTGATAGGAGCTCTAAGTAGAGTTGAGAGCCTGTTAACTAGACACGAGTAACGAGGTTAGCCTGTACGAGATATCGGGCTATAGTGCGGACACGATTGCACCATTTCTGGTGCAACGAAGGTGAGCATGTATGGACAGGTCAGTGTGACTCAAGTCGATAGTCCAAGTAGGTTATCGACTCGCATAGCTCAATGACTGTCATCGCCAGAGTATCTAGGTGTCTACCTCACGAATCGCGTCGTTACATTTCTGTAACGCTCATACCGTGCTGATCTATGGGACACGTCGCTTATTCTTGGGTCATGACAGTTGCCACAAACAAGGCACGACCTCACACCTGCGAACTTCAAGCGTTAGGCTGACGTTACATGCTTGCGTGCACTGATTCGTTTCCGAATCAGAGACCTACGAAGCCAGAGTTCGTTCACTATCATAAGTGCACTGATGCATTTGTGCCAACATTGAAGGCATCGAGATAAACAGCCGTCTTAATCAAGTGAGCACCTGAGATCAGCATGATTCGTCTATTTCTAGACGAATCAACTTCCATTCAGGTGCCTTGCTACTTAAGACGGGATTAACTCTCGATGCAACGTGCATTGGCACAACTCGTGATGTGCACTTTCACACTGGAACGAACTCTGGCTTCGTAGGTCTGTTTGTCATTTCTGACAAACTGCACGCACTGTTAGTACGTCAGCCACTTAACCGAAGTTCGTCATAAGTAGGTCGTGCGACTACGATGGCAACTTCTACTTACCAAGAATAAGCGACGTGTCCCATAATGGAAGTCGGTATGAGGTATGACTTTCGTCATACACGCGATTCCACAATGTGACACCTAACGTTTGAGGCGATGACCTGATACAAGCTATGCATGGTTCAAACCTACTTGGACTATCGACTTGAGATGATAGTACCTGTCCAACTAACAGCACCTTCGATACCTCGTTTCCGAGGTATTCGTGTCCTGTGTCAGGCCCGATATTAATGTGTGGCTAACCCTTAGGAACGTGTCTAGTTAACAGGCTCTCAACGTCATGACGAGCTCCTAGTAGCAAGCGTACGATACATTGTGACTGGTGTCTACTGGATTTCTCCAGTAACCCGACTCCGACTACAAAGTCCTGACTCATTCACCTCTGCGTGGTCCCTTGTCAGTTGAGTCGATGGTAAGTCAATGCATCAGGAATCGTGGTTAAGTCTTGTCGATCTGACACACTACTCCGCTGTCCTGTTTCCAGGACAGACGTGCATTAGCAGTTGTGGAATCATCAGTACAGTGACGAGTCGTTACTGTACGTCAGCTTGTTTGCGACTTGCAGTTAATCGACTGAGGGTCAAACGTGTCTGGTGTGTAGTCGGACTATGTGACGTTCATTTCTGAACGTACCGGCTTAGTCAACACTCCGTTGATGAGTATGACACGAACGAGTCATTGGCTCTTCGCTTCAATGTAGCACTGAACTTATGATGTTTCATACACATTACGCTCAGCGAACTGCTATGGCTAGTGTTCGGAT CC

RNA Nanostructure Sequence for One Embodiment of NR₂

(SEQ ID NO: 9) 5′-CACGAACUCAUCCUCACGCCGCCGGUCGCGUGCGGCGCCGGCAGAAGGACCUGAUGCAUCGAGCGGUGACAGCGCCACCGGAGUAGCUCAGUUAUUCGAUUCAGUAUAUUACGUAAUAUACGGCGAGAAGUUGCAGCAGUGCGCUGUAUUUUAGCAUGUCGCGCUAACUACGAUGAUUGGCAAUAAGAUUACGACCGCUAUCUCAUGGCUUAUCAAAGUGGGCAAGGUCUUGCCCACGGCGUUGACGUCCGACCCGUCGUUAAGUGUCAGAGUCGGUGUACCCUUAGAUCUGGCCACGCACGAGGUCCACCAUGGGUACUCGCGUCAGGACAGUAACCUUACUGUCGCCGCUCCGAGCGCAUAUUUCGCCUCAGGCGGGCGGUUACCCGUUAAUCUUUAGGCAAACUACGCACGGGCUUGUUUUGUGUCUCCACUCUCGGGGUACUUUGGAGUACCCCGGUAGAAGAAUGUUCCGGUUCGCGUGCGGGCGGCCGUUACACUGGGCGACUGCAUAGGGCCGCCCGCCCGCCUCGCCCUAAAUUUCACCCAUUAAGCGAGAAAGCAUUCUCGCAACUGGGUAUGGACUUGGGGUGUCCUGGAUUCGAUCACGCACCGGUCGCCGAAGUGGCGGGAAGAGCACCCCUACGUCGGGUCUUACAGGACACGCGACCCUACUUCGGCGUACCCCCCGGUACGCCCGCGCCCGCAGAGCGGUCGAUUGGUCACACGGUGGAAGUGAGUCUGUUGUCGCCGUUUCUACAAUCCGUCUGCUGCAACGGGCCAGGUUGGUUAUUAAUAACCAAAGUGCCGGAGCUCCGGCCCGUCAUAGGUGAUCCACAGCGGGUAAUAUCUUACCUGCUCCUUCAGAGGGCGGGGCAAGGGCGUCGGACGAGUGUAUUUGUUACGUCAUAACGUAACACUACGGCAUCGGGUGCUAGCGCCAGACCGCGCGCACUGGCGGCGAGAGAGUCUUAUACGCCAGGUGCUAGCCAAUAUGCGCUCACUUAAGGUGUUCAUGUACAUGAACAAAAGCUACAUGAGGACGGCUGCUUGAUUUCCAACAGGCUACGGUGUUUCUGGCUGGCCAAAUAAGUCGAGGAGGCGGGAACAUUUGACGACUCUCUGGUAUCCUUACCAGAGGGGUGAACUAACCAAAUUCCUGGAAGAAUUGGUCCUAUAGCCCUAUGAGCUAGGAACGGUCUGUUUUUCCGAGACCGUCAAGUCCAGUUGGCGGAUGGUGGGCGUCCCCACCAUGUAGGCUACCUUGUCGUGACCGAAUUCCGGCCCAGAGAAUUGGGGCAACGGCGCACUUCCUUCUGCCGCCCUCCCCGCUCUGCAGGCGCCACAGCUCGCGCACGAGCUGUCCCUACGCCAGAAGGGCUCCUCAUGCGAAGUACACCGUCAGUAAGUCUGGCCCUCUAAAAUAAAUACCUUGCCGGAGCGAGGCACUGGGAACGUAGGGACGUUCCUCUAAGUAUGCACAAUCCACAUAAGCCGACGAAUGCGCCGCACUGACGUGAAACUGGACUUGGCUGACACUCAAAAGGCCGCACCCGAGGCCCCCCGAUGUGGUCACGACCACAUCUCGCGCCGUUGUAAGGUCGAGCCCUUCAACUAAUAGUUUUAAACCCACUCGGUAGGGUUCCCUAUAACCCAAAACCCCGCCUGUUUGGGCGCGUCCUCAUAGGUGCCUAGGCAAGGCCGCUUGCCUUGGUUGCGGGGCAGAUUUUCCUAACUAAUCGUCCACCGCGAAGGCUCCCUGAAAAGCACCCGCCCGCACACGUGUAUUUGGUUAUGGUCUUCUACGAUUGGACGCAAUCGUAUCCUACCCCGUCGGGGCCGGCCCGCGACAACAGGUGAACGACCUUGGCUUGGGCAUGUCAUCGGACGGAUAGCAAGCACCGAAUCCACAACCUGUCGACAAGGCUGAUCCUCCCUCCGGGGAGGAUCAGUGGGCAAUACAGGUUGUUAGUCCGGUGCUUGCUCGCUCGAUAAGGUCGUACCGUGUGUGUCGCGGGCCCUUCUGGCCGGGGUAGGAUCAGCACCAAAUGGUGCUGAGAAGACCAUUAAAGGGUACACGUGUCAACAUAGAUAGGAUUUUACGCCGGGUGCUUUUGUAGUUAGCUUCGCGGUGGAUCACCAGUUAGGAGUCGUGGAUUGGCAACCACAUUAACGACGGUUAAUGAGGCACCUAAAUCAUUUCGCCCAAAUCUACCAAGUUUUGGGUUAAGGGUAACCCUACCCGCGCGGUUUAAAACGACCUUACAACGGCGCGAACGCCAUGUACGCAUGGCGUGGGGGGCCGACCGAAUGGCCUUUUGGGGUAUGCCCAAGUCCAGAUUAACGUCAGUGCGUUGGAAAUGUCGGCUUAUAGCAAAUCUGCAUACUUAGAGUGACGCUAGAGCGUCACCAGUGCCUCUGAUUCCUAAGGUAUUCCUCAGCCUCCCUCUAACUAUGCACAGGGCCAGAUCGCCCAGCGGUGUACACCAAUUCAGGAGCCGGCCCCGAGUAGGGUGCAGAUGUGGGCAUCUGCAGGCGCCUGGGUCGGCUGGAGGGCGGGUGUCGAAAGUGCGCCGGCGACCGAAUUCUCUGGGCCGGAAUUAACGGUUUCCCGAGUGGCGGUCAAUUGCCCAUAGCCUACAGCGACACGCAGGUGUCGCUCCGCCAACGAAUUAGUACGGUCUCUCCGGUGGAGACCGUUCCUACAGACUCGGGCUAUAGGUUCGCAUGUUCCAGGAACCCUUUAAGUUCACCCCUGCACCGAGGCCGGUGCAGAGUCGUCAGACGGGCGCGCCUCCUCCUUAUUGCUGGCCAGCUCCGGUAAGAUUAGCCUGGCGCAUUCCAAGCAGCAAAUGAUUUGUAGCUUUAAUUAAGGCACCUUAAUUCCUUAAGUGAUCGUCUCUUGGCUAGUCGUGCGUGUAUAAGACUCUCUCGCCGCCAGUGGAGUGGGUCUGGCGCUAAUUCGGUCUGCCGUAGGUUCCUACCUUUGUAGGAACAAUACACUCUAACUAGGCCCUUGCCUGCGUAGUUGAAGGAGCAGUUCAGAAACAACCCGCUGUGGACGAUUUAUGACGGAGGAAUCAUCCGGCACUUGCUUUAUCGAUAAAGCACCUGGCCCGUUACGCAGGACGGAUUCGGGCGGCGGCGACAAGCUCAUAACUUCCUCACCUGUACCAAUCGAAGCCGACCCGGGCGCGUCAGCGGGGGUACCCGCUGAGAAGUAGGGAUGGGCGUCCUGGGUGCUCUUCCCGCCACUUCGUUGCCCCGUGCGUGAUCGGACUAAGGACACCCACUAAUUCUACCCAGUUCGUAACGAUAUCGUUACGUUAAUGGGUGAAAUAACUGGCGAGGCGGGGUAGAAACCCUAUGCAGCUUACUGAUGUAACGGCCGGCGUGACGCGAACCCGCCCGUCCUUCUACCGUGAUUGGUGUCCCAAUCACGAGAGUGGCAUCAUGAGAUAAGCCCGCCGCCCUCUUGCCUAAAGUUUCACGGGUAACCGUUGGUAGAAGGCGAAAGAGACGAUCGGAGCGGCCCCCGUCGCCUGACGGGGCUGACGCGAGUACCCAUGGUCGCAUGCCCGUGCAGAUGGACCCACCUGGCGGCCAGAUCUAUAGGGACACCGACUGCAUACCCUAACGACGGCUAGUUAGUCAACGCCGGGGCCGCACACGCGGCCCCUUUGAUAAGCGACAGACACAAGGUCGUAAUGACUUAUUCAAUCAUCCAGGGAGCCGCGACAUGGUGCAAGGCAGCGCACCUGCGUAACUUCUCGCCCGAGGAUACUCAUCCUCGUGAAUCGAAUUUAGGAGCUACGGAAAAACCGCUGUCACAUCCGGCCGCAUCAGGUUCGACACCCGGCGCCGCGCCCAUCCGGCGGCGUGAGGAUGAGUUCG UGUCCGCGGGCCACC-3′

DNA Sequence Encoding RNA Nanostructure Having SEQ ID NO: 9

(SEQ ID NO: 120) 5′-CATGAACTCATCCTCACGCCGCCGGTCGCGTGCGGCGCCGGCAGAAGGACCTGATGCATCGAGCGGTGACAGCGCCACCGGAGTAGCTCAGTTATTCGATTCAGTATATTACGTAATATACGGCGAGAAGTTGCAGCAGTGCGCTGTATTTTAGCATGTCGCGCTAACTACGATGATTGGCAATAAGATTACGACCGCTATCTCATGGCTTATCAAAGTGGGCAAGGTCTTGCCCACGGCGTTGACGTCCGACCCGTCGTTAAGTGTCAGAGTCGGTGTACCCTTAGATCTGGCCACGCACGAGGTCCACCATGGGTACTCGCGTCAGGACAGTAACCTTACTGTCGCCGCTCCGAGCGCATATTTCGCCTCAGGCGGGCGGTTACCCGTTAATCTTTAGGCAAACTACGCACGGGCTTGTTTTGTGTCTCCACTCTCGGGGTACTTTGGAGTACCCCGGTAGAAGAATGTTCCGGTTCGCGTGCGGGCGGCCGTTACACTGGGCGACTGCATAGGGCCGCCCGCCCGCCTCGCCCTAAATTTCACCCATTAAGCGAGAAAGCATTCTCGCAACTGGGTATGGACTTGGGGTGTCCTGGATTCGATCACGCACCGGTCGCCGAAGTGGCGGGAAGAGCACCCCTACGTCGGGTCTTACAGGACACGCGACCCTACTTCGGCGTACCCCCCGGTACGCCCGCGCCCGCAGAGCGGTCGATTGGTCACACGGTGGAAGTGAGTCTGTTGTCGCCGTTTCTACAATCCGTCTGCTGCAACGGGCCAGGTTGGTTATTAATAACCAAAGTGCCGGAGCTCCGGCCCGTCATAGGTGATCCACAGCGGGTAATATCTTACCTGCTCCTTCAGAGGGCGGGGCAAGGGCGTCGGACGAGTGTATTTGTTACGTCATAACGTAACACTACGGCATCGGGTGCTAGCGCCAGACCGCGCGCACTGGCGGCGAGAGAGTCTTATACGCCAGGTGCTAGCCAATATGCGCTCACTTAAGGTGTTCATGTACATGAACAAAAGCTACATGAGGACGGCTGCTTGATTTCCAACAGGCTACGGTGTTTCTGGCTGGCCAAATAAGTCGAGGAGGCGGGAACATTTGACGACTCTCTGGTATCCTTACCAGAGGGGTGAACTAACCAAATTCCTGGAAGAATTGGTCCTATAGCCCTATGAGCTAGGAACGGTCTGTTTTTCCGAGACCGTCAAGTCCAGTTGGCGGATGGTGGGCGTCCCCACCATGTAGGCTACCTTGTCGTGACCGAATTCCGGCCCAGAGAATTGGGGCAACGGCGCACTTCCTTCTGCCGCCCTCCCCGCTCTGCAGGCGCCACAGCTCGCGCACGAGCTGTCCCTACGCCAGAAGGGCTCCTCATGCGAAGTACACCGTCAGTAAGTCTGGCCCTCTAAAATAAATACCTTGCCGGAGCGAGGCACTGGGAACGTAGGGACGTTCCTCTAAGTATGCACAATCCACATAAGCCGACGAATGCGCCGCACTGACGTGAAACTGGACTTGGCTGACACTCAAAAGGCCGCACCCGAGGCCCCCCGATGTGGTCACGACCACATCTCGCGCCGTTGTAAGGTCGAGCCCTTCAACTAATAGTTTTAAACCCACTCGGTAGGGTTCCCTATAACCCAAAACCCCGCCTGTTTGGGCGCGTCCTCATAGGTGCCTAGGCAAGGCCGCTTGCCTTGGTTGCGGGGCAGATTTTCCTAACTAATCGTCCACCGCGAAGGCTCCCTGAAAAGCACCCGCCCGCACACGTGTATTTGGTTATGGTCTTCTACGATTGGACGCAATCGTATCCTACCCCGTCGGGGCCGGCCCGCGACAACAGGTGAACGACCTTGGCTTGGGCATGTCATCGGACGGATAGCAAGCACCGAATCCACAACCTGTCGACAAGGCTGATCCTCCCTCCGGGGAGGATCAGTGGGCAATACAGGTTGTTAGTCCGGTGCTTGCTCGCTCGATAAGGTCGTACCGTGTGTGTCGCGGGCCCTTCTGGCCGGGGTAGGATCAGCACCAAATGGTGCTGAGAAGACCATTAAAGGGTACACGTGTCAACATAGATAGGATTTTACGCCGGGTGCTTTTGTAGTTAGCTTCGCGGTGGATCACCAGTTAGGAGTCGTGGATTGGCAACCACATTAACGACGGTTAATGAGGCACCTAAATCATTTCGCCCAAATCTACCAAGTTTTGGGTTAAGGGTAACCCTACCCGCGCGGTTTAAAACGACCTTACAACGGCGCGAACGCCATGTACGCATGGCGTGGGGGGCCGACCGAATGGCCTTTTGGGGTATGCCCAAGTCCAGATTAACGTCAGTGCGTTGGAAATGTCGGCTTATAGCAAATCTGCATACTTAGAGTGACGCTAGAGCGTCACCAGTGCCTCTGATTCCTAAGGTATTCCTCAGCCTCCCTCTAACTATGCACAGGGCCAGATCGCCCAGCGGTGTACACCAATTCAGGAGCCGGCCCCGAGTAGGGTGCAGATGTGGGCATCTGCAGGCGCCTGGGTCGGCTGGAGGGCGGGTGTCGAAAGTGCGCCGGCGACCGAATTCTCTGGGCCGGAATTAACGGTTTCCCGAGTGGCGGTCAATTGCCCATAGCCTACAGCGACACGCAGGTGTCGCTCCGCCAACGAATTAGTACGGTCTCTCCGGTGGAGACCGTTCCTACAGACTCGGGCTATAGGTTCGCATGTTCCAGGAACCCTTTAAGTTCACCCCTGCACCGAGGCCGGTGCAGAGTCGTCAGACGGGCGCGCCTCCTCCTTATTGCTGGCCAGCTCCGGTAAGATTAGCCTGGCGCATTCCAAGCAGCAAATGATTTGTAGCTTTAATTAAGGCACCTTAATTCCTTAAGTGATCGTCTCTTGGCTAGTCGTGCGTGTATAAGACTCTCTCGCCGCCAGTGGAGTGGGTCTGGCGCTAATTCGGTCTGCCGTAGGTTCCTACCTTTGTAGGAACAATACACTCTAACTAGGCCCTTGCCTGCGTAGTTGAAGGAGCAGTTCAGAAACAACCCGCTGTGGACGATTTATGACGGAGGAATCATCCGGCACTTGCTTTATCGATAAAGCACCTGGCCCGTTACGCAGGACGGATTCGGGCGGCGGCGACAAGCTCATAACTTCCTCACCTGTACCAATCGAAGCCGACCCGGGCGCGTCAGCGGGGGTACCCGCTGAGAAGTAGGGATGGGCGTCCTGGGTGCTCTTCCCGCCACTTCGTTGCCCCGTGCGTGATCGGACTAAGGACACCCACTAATTCTACCCAGTTCGTAACGATATCGTTACGTTAATGGGTGAAATAACTGGCGAGGCGGGGTAGAAACCCTATGCAGCTTACTGATGTAACGGCCGGCGTGACGCGAACCCGCCCGTCCTTCTACCGTGATTGGTGTCCCAATCACGAGAGTGGCATCATGAGATAAGCCCGCCGCCCTCTTGCCTAAAGTTTCACGGGTAACCGTTGGTAGAAGGCGAAAGAGACGATCGGAGCGGCCCCCGTCGCCTGACGGGGCTGACGCGAGTACCCATGGTCGCATGCCCGTGCAGATGGACCCACCTGGCGGCCAGATCTATAGGGACACCGACTGCATACCCTAACGACGGCTAGTTAGTCAACGCCGGGGCCGCACACGCGGCCCCTTTGATAAGCGACAGACACAAGGTCGTAATGACTTATTCAATCATCCAGGGAGCCGCGACATGGTGCAAGGCAGCGCACCTGCGTAACTTCTCGCCCGAGGATACTCATCCTCGTGAATCGAATTTAGGAGCTACGGAAAAACCGCTGTCACATCCGGCCGCATCAGGTTCGACACCCGGCGCCGCGCCCATCCGGCGGCGTGAGGATGAGTTCG TGTCCGCGGGCCACC-3′

Example 2

TLR3 and TLR7/8 HEK-293T reporter lines were used to study whetherRNA-origami could activate TLR3-signaling pathway and/or the TLR7pathway. The results indicated that the RNA-origami activateTLR3-signaling pathway, but not the TLR7. Unlike dsRNA-mediatedactivation, the stimulatory activity observed was independent oftransfection, which suggests that RNA-origami are taken up by HEK-293Tcells to trigger TLR3-signaling pathway, rather than mediated throughcytoplasmic RNA sensors, i.e., MDA5/RIG. Interestingly, although theRNA-origami and polyIC displayed a comparable level of activation inTLR3-reporter line, much more potent activation of splenocytes was foundby RNA-origami than polyIC (see FIG. 3 and FIG. 4). This findingsuggests that antigen presenting cells present in the spleen can uptakeRNA-origami for the activation of these immune cells.

Furthermore, the cytokine profiles were examined in mice receivingintraperitoneal injection of RNA origami or low molecular weight polyICthat is in the same size range as the present RNA-origami.Interestingly, it was found that the cytokine profile in RNA-origamimice showed high levels of IL12, chemokines, but low and moderate levelsof TNFa and IL6, respectively (FIG. 12). PolyIC used in this example haslow molecular weight, whereas the one used in Takeda's report likely arehigh molecular weight PolyIC, which is associated with high toxicity.(Takeda et al., A TLR3-Specific Adjuvant Relieves Innate Resistance toPD-L1 Blockade without Cytokine Toxicity in Tumor Vaccine Immunotherapy,Cell Rep. 2017 May 30; 19(9):1874-1887.) Nevertheless, the polyIC-LMWdid not induce significant elevation of these cytokines, similar to thestudy reported by Zhou, Y., 2012. TLR3 activation efficiency by high orlow molecular mass polyIC. Innate Immunity. 19:184-192, which shows thathigh molecular weight (HMW) PolyIC (also referred to as “PolyIC-HMW”) ismore potent in vivo than low molecular weight (LMW) polyIC (polyIC-LMW).In addition, PolyIC-HMW is usually used as vaccination adjuvants and itssystemic application is associated with toxicity. Compared to the levelsof TNFa and IL-6 shown in Takeda's study, the levels of these cytokinesinduced by RNA-origami are at the range of those induced by two ARNAX,i.e., have low toxicity. Thus, the present RNA-origami may function morelike ARNAX. On the other hand, elevation of three chemokines, CXCL9,CXCL10 and CCL2 play important roles to recruit CD8-T and NK cells tomount anti-tumor immunity.

To determine whether the in vitro stimulation of immune cells can betranslated into anti-cancer immune adjuvants, CT26 peritoneal coloncarcinoma model was used, which has been explored as a peritonealmetastatic model, to test whether RNA-origami can reduce tumor growth inthe peritoneal cavity. To monitor tumor growth in real time, a gene iRFPwas introduced into CT26 cells, which codes for a near infraredfluorescence protein, such that the growth of tumor cells is measured byiRFP fluorescence intensity. A higher fluorescence intensity isindicative of a larger tumor mass. Specifically, on day 0, mice receivedone million CT26-iRFP cells via i.p. injection. The mice were treatedwith RNA-origami or control PBS on day 1, 3 and 7 at 16 microgram/dose,and tumor cells in peritoneal cavity was monitored by iRFP fluorescenceintensity using LI-COR Pearl Small Animal Imaging System. It was foundthat while the mice injected with PBS developed tumor quickly (with10-12 days), the mice treated with RNA-origami showed a significantreduction in tumor growth (FIG. 13). Thus, at a rather low doses used inthe experiment, RNA-origami suppressed tumor growth. When the cytokinesproduced from ascites fluid that were accumulated within tumor cellspresent in the peritoneal cavity were analyzed, it was found that theascites contained very high levels of immunosuppressive cytokines,including TGFb1, TGFb2, IL-10 and IL-4 (FIG. 14). In contrast, for thetumor-bearing mice treated with RNA-origami, they had much lower levelsof immunosuppressive cytokines, but elevated levels of anti-tumorproinflammatory cytokines, which correlates with the small tumor load inthe treated mice.

Example 3

Induction of Strong Anti-Tumor Immunity by RNA-Origami Complexed withTumor Penetrating Peptide (TPP)

Nucleic acid based Toll-like receptor ligands, such as poly IC, ssRNAand CpG oligonucleotides are potent adjuvants via activation of TLR3,TLR7/8 and TLR9 signaling pathways, respectively. Tumor-specificantigens in combination with these TLR ligands have been explored ascancer vaccines to reduce tumor growth. Building on the finding of RNAorigami as a TLR3 ligand discussed above, peptide-tagged RNA-origamicomplexes were constructed, and the complexes were shown to be stableand able to induce strong anti-tumor immunity.

Heat shock protein 70 (HSP70) is a cellular stress response protein,presumably protecting cells from toxic agents and harsh environment. Onthe other hand, because of its chaperon function in associated withtumor specific or tumor-associated antigens (TSAs or TAAs), HSP70 hasalso been explored as a TAA. It was reported to induce multifacetedresponses against cancer cells, including both innate and adaptiveimmunity. Interestingly, one peptide derived from the C-terminus ofHSP70, also referred to as “TKD peptide”, has been demonstrated (1) toactivate NK cells, (2) to direct tumor-targeted binding andinternalization, and (3) to promote DC cross-presentation and ultimatelyinduction of cytotoxic T cell responses toward tumor cells. It wasinvestigated whether the combination of this peptide with RNA-origamiwould constitute a potent cancer vaccine.

Given the potent and unique adjuvant activity of RNA-origami, it washypothesized that complexing RNA-origami with TKD peptide would increasetumor-specific immunity. RNA-origami was complexed with tumor targetingpeptide (TPP) TKD-peptide. TKD (TPP)-peptide has the sequenceTKDNNLLGRFELSG (SEQ ID NO: 121) (C-terminal region of human HSP70),which is highly homologous to murine HSP70 sequence TRDNNLLGRFELSG (SEQID NO: 122).

To simplify the complex formation with RNA-origami, the TKD was modifiedby adding a cystine (C) at the N-terminus and adding 10 lysine residuesto the C-terminus of the TKD peptide, thus creating CTKD-K10:CTKDNNLLGRFELSGGGSKKKKKKKKKK (SEQ ID NO: 3). The C residue allowspeptide-dimerization to promote peptide binding to and clustering ofHSP70 on the surface of tumor cells. Pre-incubation of CTKD-K10 withsplenocytes can activate NK cells, which in turn kill tumor cells.CTKD-K10 can also bind to many tumor cells, also referred to as tumorpenetrating peptide (TPP) and upon binding, it can induceinternalization of the peptides, possibly via HSP70 oligomerization,reaching to endosome, lysosome and even mitochondria.

RNA-origami are negatively charged structure, so the positive charge ofpolylysine on the TKD-K peptides enables direct, non-covalent complexformation with the RNA-origami. The complex was demonstrated formationby gel electrophoresis (FIG. 16). Depending on the RNA:peptide ratios,the size of the complexes is increased and some become aggregated.Different RNA-OG/TTP ratios lead to different sizes of complexes. Thecomplex appears stable after its formation as the old and new complexesformed at 1:200 ratios displayed similar pattern of mobility (FIG. 16,lane 3 and lane 7).

Different complexes exhibit different binding/internalization profiles,as shown by flow cytometry (FIG. 17). It was observed that theinternalization of RNA-peptide complex is hindered if more peptidesassociated with the RNA. It was found that at RNA:peptide ratio of 1:100or 1:200, the complex size was slightly shifted up, but is still takenup by both CT-26 colon cancer cell line and RAW-264 macrophage line(FIG. 17). Higher internalization of RNA-origami (OG) by RAW cells thanCT-26. Upon increase amount of the peptide, the lower level of bindingto both CT-26 and RAW cells. It was predicted that the combination ofthe RNA-origami and TKD peptide would further enhance and integrate TLR3activation, NK-activation, antigen-cross presentation for effectiveinduction of cytotoxic T cell responses.

In an in vivo tumor model, the RNA-peptide complex was tested at the1:100 ratio. Interestingly, a single injection of this complex into amouse-bearing tumor led to complete tumor regression (FIG. 18).Red-fluorescence positive tumor cells were inoculated at day 0 and tumornodule formed on day 9 (i.e., pre-treatment). These mice were thentreated with a single injection of different types of RNA structures,free RNA or RNA-origami coated with tumor-targeting peptide (TTP). Themice were monitored for more than 20 days, and tumor regression wasfound in the mouse receiving the RNA-Origami polymer, but not othergroups (including RNA-origami only group).

In a separate experiment, RNA-peptide complex (1:200 ratio) were alsoinjected intraperitoneally, where the intraperitoneal colon tumor cellswere inoculated (FIG. 19A). One RNA-OG/TPP and RNA-origami (OG) out offive treated mice showed tumor regression, whereas all the controlgroups, including the mice receiving free RNA, succumbed to tumorgrowth. The adaptive immunity of splenocytes recovered from thetumor-free mouse treated with RNA-OG/TPP were further tested and it wasfound that these cells are reactivated in vitro by the co-culture withTPP, but not when administered irrelevant KLH peptides (FIG. 19B-FIG.19C). Thus, tumor-targeted adaptive immunity was elicited by theRNA-OG-TPP complexes.

Example 4

Incorporation of Modified Ribonucleotides into Transcribed ssRNASequence

The same RNA strand synthesis procedure as above was performed but amodified ribonucleotide was added during the in vitro transcriptionreaction. The 5-Aminoallyluridine-5′-Triphosphate (AA-UTP) iscommercially available and is a good substrate for RNA polymerases toeasily incorporate into RNA strand. The ratio of AA-UTP to UTP(non-modified ribonucleotides) was varied from 1:10 to 1:50, whichgenerated 2-10% AA-UTP incorporation into the RNA strand, respectively.

The amine group in the resulting synthesized RNA strand is capable ofconjugatation with peptides or antibodies through a bifunctionalcompound (e.g., SMCC crosslinker or sulfo-SMCC). When the total lengthof the ssRNA strand is 1000 bp, about 20 to about 100 AA-UTP areincorporated, representing potentially about 20 to about 100 conjugationsites.

Example 5

Stability of Peptide-ssRNA Complexes (NR₁—R₃, Wherein R₃ is a Peptide)(“RNA-OG”)

The ssRNA from Example 1 was synthesized. Two different peptides(“Pep-1” and “Pep-2”) that each comprise an additional 10 lysineresidues were added to a solution of the rectangular sheet scaffoldformed from folded ssRNA in 1×PBS buffer and allowed to form the complexformation between peptide and the RNA-OG. Similar complexes wereconstructed using the non-foldable polyIC chain. The peptides were addedat molar ratios of (none), 1:100 (ssRNA:peptide), and 1:200 (ssRNApeptide). The RNA-OG or polyIC along with their peptide complexes wererun on non-denaturing agarose gel electrophoresis. The left-most lanerepresents no added peptide. The right-most lane is a 1 Kb marker. Theresults show that for the ssRNA:peptide complexes were soluble andstable in solution, and the total MW increased in a dose-dependentmanner as the peptide amount was increased. The polyIC control, however,failed to exhibit any form of soluble or stable complex under the sameconditions.

Example 6

Demonstration of pH Sensitivity of Linker

In certain embodiments, the linker is pH-sensitive. The RNA duplexlinked to double nanobots can be responsive to an acidic environment forbeing dehybridized. RNA duplex stability is pH sensitive as a lower pHreduces the melting temperature of short RNA-duplexes. Thedouble-nanorobot is incubated in an acid low pH cell culture condition(pH 6.8) used to mimic tumor microenvironment. RNA-duplexes that arestable under pH7.4, but labile at pH 6.8, are designed using the Tiamatsoftware and are tested under such denaturing conditions. Factorsaffecting the pH sensitivity include the hybridization length, the GCcontent of the sequences, and the proximity of the GC content to theterminae of the hybridized sequence.

In a further experiment, serum that contains nucleases are added to theculture to facilitate the degradation of RNA-duplexes to dissociate thetwo RNA nanostructures whereas the RNA-cage or RNA-origami are likelyresistant to the nuclease.

Example 7

Thrombin Loading and Release

To the prepared RNA nanostructure is further added a 20-fold molarexcess of fasteners and a fivefold molar excess of targeting strands,including the AS1411 sequence. Thrombin molecules are loaded on the topor the bottom surface of RNA nanostructure scaffolds, wherein thescaffold is an origami sheet. After fastening of the rectangular sheetinto a tube, the top surface is rolled inside the tube due to curvaturedriving forces. Thus, thrombin is loaded on the inside or the outsidesurface of origami tubes. Thrombin loaded inside tubes is protected andshielded before delivery to the target location in vivo, while uncagedthrombin is subject to degradation. The topography is assayed through aplatelet aggregation assay. For the rectangular RNA scaffold sheetstructures, the ssRNA strand and thrombin-loading strands are mixed in1×TAE-Mg buffer. The mixtures are then placed on an Eppendorf thermalcycler with the program: rapid heating to 65° C., then cooling to 25° C.at a rate of 10 min/° C. for annealing. For a control rectangular sheetstructures, ssRNA and thrombin-loading strands are mixed together andannealed. Thrombin-RNA conjugates are then mixed with the rectangularRNA nanostructure scaffold sheet or control nanostructure. The mixturesare heated to 45° C. and cooled to 25° C. at a rate of 10 min/° C. tofacilitate annealing.

The resultant thrombin-rectangle-RNA nanostructure origami assembliesare purified using 100 kD centrifugal filters to remove excessthrombin-RNA nanostructure conjugates. After loading thrombin, a 20-foldmolar excess of fastener strand polynucleic acids and a 5-fold molarexcess of targeting strand polynucleic acids, are added to induce theformation of tube structures. To facilitate annealing, the mixture isheated to 37° C. and then cooled to 15° C. at a rate of 10 min/° C.

The thrombin-loaded rectangular and tubular RNA nanostructure origaminanostructures are applied to the platelet aggregation assay describedherein. Additionally, thrombin-RNA nanostructure and control RNAnanostructure are pretreated with proteinase K for 15 min at roomtemperature to remove the thrombin molecules on the outside surface ofthe tubes. Next, the proteinase-treated nanostructures are degraded with20 U/ml DNase I (Invitrogen, Carlsbad, Calif., USA) at 37° C. for 30 minto expose the thrombin molecules inside the tubes. The expectedconsiderably low platelet aggregation using thrombin-loaded RNAnanostructure or RNA nanostructure controls without such treatmentindicates that only a small amount of thrombin molecules are loaded ontothe outside surface of the tubes. Aggregation results of tube structuresare obtained after proteinase and DNase I treatment and furtherdemonstrate potent platelet aggregation, reflecting the vast majority ofthrombin are loaded inside the RNA nanostructure tubes. The results ofthese experiments are used to estimate the percentage of thrombin loadedon the inside and outside surfaces of the thrombin-loaded RNAnanostructures. These results will demonstrate that the thrombinmolecules can be arranged inside the RNA nanostructure in a shieldedstate by design.

Example 8

Confirming the Functionality of the IFN-γ Lock

The IFN-based RNA cage efficacy in a model tumor environment isdemonstrated.

A RNA nanostructure comprising the sequence of SEQ ID NO: 9 is made bythe methods described above. As shown in FIG. 38, the RNA nanostructurewill exhibit a tube configuration in a closed RNA cage state when theIFN-γ lock is outside of a high-IFN concentration tumor environment. Insome embodiments, high-IFN concentrations are greater than 5 μg/mL. Themixture is heated to 37° C., then cooled to 15° C. at a rate of 10 min/°C. to promote assembly. The RNA nanostructure further comprises ananti-PD1 antibody as a moiety internal to the enclosed RNA cage. The RNAnanostructure is linked with an IFN-γ-binding DNA aptamer with highbinding affinity.

A 34-mer IFN-γ-binding aptamer sequence is used for the studies (IDTTechnologies, San Diego, Calif.):5′-NH2-C6GGGGTTGGTTGTGTTGGGTGTTGTGTCCAACCCC-C3-SH-3′ (SEQ ID NO: 123).The aptamer is modified at the 5′-terminus with a C6-disulfide[HO(CH2)6-S-S-(CH2)6-] linker to bind the 5′ terminus to a portion ofthe RNA nanostructure. In some embodiments, the unmodified aptamersequence is extended with additional sequences to allow its assemblyinto the RNA nanostructure and lock the two ends of the RNA sheet.

The effector molecules entrapped within the RNA nanocage is an anti-PD1antibody, anti-PDL-1 antibody, PD1-binding aptamers or PDL1-bindingaptamers. The selected antibody will be conjugated to the amine-modifiedUTP designed to be positioned within the nanocage. Alternatively, theanchor sequences that are incorporated into the RNA-nanostructures areused to attach selected aptamers.

The RNA-nanocage is opened upon interaction with IFN-γ. For that, theassembled RNA-nanocage is incubated with various concentrations of IFN-γand nonspecific protein control (IgG, BSA, etc.) and concentrationdependent RNA nanocage opening is observed. The concentration of IFN-γthat opens the RNA nanocage is assessed by both gel electrophoresisand/or AFM. To further determine whether the enclosed effector moleculesare accessible and function to bind their targets, a sandwich ELISAassay is used to detect the access of these molecules to the PD1 orPDL-1 coated to the ELISA plate and the molecules are detected byfluorescence-labeled PD1 or PDL1. For this test, the originalRNA-nanocage in the absence of IFN-γ is included as a negative controlsince the antibodies or aptamers are kept inside the cage and notexposed. Once the cage is opened, effector molecules such as checkpointinhibitor antagonists do not have to be released from RNA-nanostructuresto antagonize checkpoint protein inhibitory pathways, because they canstill function to bind and block the checkpoint molecules that areexpressed on the cell surface of PD1+ or PDL-1+ cells.

Example 9

The Efficacy of RNA Double Robot Nanostructure

The RNA nanostructure of Examples 1 and 8 will be separately preparedand linked by cross-hybridization. The RNA-nanostructure of Example 1(NR₁) stimulates splenocytes as demonstrated in Example 1, and theRNA-nanostructure of Example 8 (NR₂) is further linked with a anti-PD-1antibody checkpoint inhibitor antagonist to demonstrate the blockingcheckpoint effect. The PD1/PDL-1 bioassay kit (Promega, protocolattached) is used to assess the functional activity of the effectormolecules (checkpoint inhibitor antagonists) that become accessible tothe cells. The results demonstrate that upon presentation to a tumorcell, the RNA nanostructure of Example 8 in a closed cage open in thepresence of localized high concentrations of IFN-γ to unlock the cage,thereby releasing active anti-PD1 antibody which blocks the checkpointpathway.

Example 10

In Vivo Efficacy of RNA Double Robot Nanostructure

Additional experiments demonstrate this method works in vivo in theperitoneal tumor environment. To assess the in vivo efficacy of thenanorobots, nude mice bearing 100 mm³ MDA-MB-231 tumors are randomlydivided into six groups of eight mice per treatment group and aretreated with saline, free thrombin, empty double nanorobot, nontargetedRNA nanostructure double robot comprising thrombin (free of targetstrands) made by the methods described herein, targeted RNAnanostructure comprising thrombin (comprising target strands), (1.5 Uaccumulated thrombin/mouse), by tail vein injection every 3 d for atotal of six treatments. The day of the first injection is designatedday 0. Tumors are measured with calipers in three dimensions.

The following formula is used to calculate tumor volume:Volume=(length×width²)/2.

To confirm antitumor efficacy, a syngeneic B16-F10 melanoma tumor modelis established by subcutaneous injection of 5×10⁶ murine B16-F10 cellsinto the right posterior flank of C57BL/6J mice. When the tumors reach asize of 150 mm³, the mice (ten mice per group) are treated intravenouslywith the cohort treatments as described above, every other day for 14 d.Tumor volume is determined as described above. The animals areeuthanized after the last treatment, and the livers are excised andweighed. Liver sections are stained with H&E for metastasis analysis.

Two other tumor models, an ovarian cancer SK-OV3 xenograft model and aninducible KrasG12D lung tumor model are used to investigate theversatility of the thrombin-comprising RNA nanostructure double robot.For the SK-OV3 model, nude mice bearing 100 mm³ SKOV3 xenografts (eightmice per group) are treated intravenously with the cohort treatments asdescribed above, with an additional cohort treated with a scrambledaptamer control, periodically for a total of 6 treatments (˜1.5 Uaccumulated thrombin/mouse). The inducible KrasG12D mice are fed withdoxycycline diet since the 6^(th) week after birth to induce primarylung adenomas. After being induced for 2 weeks, mice with tumors arerandomly divided into four groups (three animals per group) and treatedwith the cohort treatments as described above by intravenous injectionperiodically. The progress of lung tumors is monitored by MR imaging 1week and 2 weeks after treatment started. The results demonstrate theefficacy of the RNA nanostructure double robot comprising thrombin overthe controls.

In Vivo MR Imaging.

TetO-KrasG12D transgenic mice are imaged using a 7.0 T Bruker Biospecanimal MRI instrument (Germany). The imaging parameters are set asfollows: FOV (field of view)=3×3 cm², MTX (matrix size)=256×256, slicethickness=1 mm, TE=61.2 ms, TR=2320 ms, and NEX=4. The mice areanesthetized with 1.5% isoflurane delivered via nose cone before andduring the imaging sessions.

Cell Viability Assay.

The cytotoxicity of the RNA nanorobot structure is assessed in murineendothelial bEnd3 cells. Cells (2,000 cells/well) are added to the wellsof a 96-well plate (Corning, Woburn, Mass., USA). After culturing at 37°C. for 4 h, the cells are incubated with RNA nanorobot structure ateither 3.3 nM or 6.6 nM (in PBS) for a further 24, 48 or 72 h. Theproportion of viable cells is evaluated using a CCK-8 kit(Sigma-Aldrich, St. Louis, Mo., catalog No. 96992). Blank wells onlywith culture media and PBS-treated wells are used to define 0 and 100%viability, respectively.

Determination of Platelet Surface P-Selectin, Plasma Fibrin and ThrombinLevels and Platelet Counts.

Nude mice bearing MDA-MB-231 tumors are injected intravenously with thecohort treatments as described above periodically for a total of sixinjections. Mouse whole blood is then collected retro-orbitally into a3.8% sodium citrate solution in blood collection tubes at the indicatedtime points. For platelet activity studies, the blood is mixed with anequal volume of 2% paraformaldehyde for 30 min at RT and centrifuged toobtain platelet-rich plasma (PRP). The PRP is incubated withFITC-conjugated P-selectin-specific monoclonal antibodies and analyzedby flow cytometry. Fibrin or thrombin levels in the PRP are quantifiedby enzyme-linked immunosorbent assay (ELISA) kits (Abcam, ab108844 andab157527, respectively). Platelet numbers are counted manually with ahemocytometer using optical microscopy. The results demonstrate theefficacy of the RNA nanostructure double robot comprising thrombin overthe controls.

Example 11

DNA Cage

Methods

The design and characterization of DNA half-cages and full-cages.

DNA origami half-cage and structures were designed with caDNAno, eachused one M13mp18 ssDNA as the scaffold. Detailed design schemes areshown in FIGS. 7-9. One or both of the half-cages containedsingle-stranded probe strands (4 in each half-cage) extended toward theinside of the cage for binding with the DNA conjugated enzymes. Two ofthe half-cages can be linked together to form a fully enclosed full-cagethough 24 linker strands. To form each of the half-cages, the M13mp18ssDNA was mixed with the corresponding staples at a 1:10 molar ratio in1×TAE-Mg2+ buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA and 12.5 mMmagnesium acetate, pH 8.0), annealed from 80° C. to 4° C. for 37 h. Theexcess staple strands were removed by the filtration of the DNA cagessolution using 100-kD Amicon filter with 1×TAE-Mg2+ buffer for threetimes. To form a full-cage, 24 single-stranded DNA linkers wereincubated with the two purified half-cages at a molar ratio of 5:1 forthree hours at room temperature, in order to connect the two half-cagestogether.

Enzyme-DNA Cage Assembly.

A 15-fold molar excess of oligonucleotide-conjugated enzyme wasincubated with the DNA half-cage containing capture strands. Proteinassembly was performed using an annealing protocol in which thetemperature was gradually decreased from 37° C. to 4° C. over 2 h andthen held constant at 4° C. using an established procedure. TwoEnzyme-attached half cages were then assembled into a full cage byadding DNA linkers as described above. The DNA caged-enzymes werefurther purified by agarose gel electrophoresis to remove excess freeenzymes.

Preparation, Purification, and Characterization of Protein-DNAConjugates.

Protein-DNA conjugation-SPDP conjugation chemistry was used to coupleenzymes to oligonucleotides as reported previously. Enzymes (GOx, HRP,G6pDH, LDH, MDH and β-Gal) were first conjugated with SPDP atenzyme-to-SPDP ratios of 1:5, 1:20, 1:3, 1:5, 1:5, and 1:5,respectively, in HEPES buffer (50 mM HEPES, pH 8.5) for 1 h at roomtemperature. Different values of SPDP-to-Protein ratio were used due tothe varied number of accessible surface lysine residues for eachprotein. Excess SPDP was removed by washing with 50 mM HEPES bufferusing Amicon centrifugal filters (30 kD cutoff). The SPDP couplingefficiency was evaluated by monitoring the increase in absorbance at 343nm due to the release of pyridine-2-thione (extinction coefficient: 8080M⁻¹ cm⁻¹).

TCEP-treated thiolated DNA (/5ThioC6-/-TTTTTCCCTCCCTCC (SEQ ID NO: 126)(P), or/5ThioC6-D/-TTTTTGGCTGGCTGG (SEQ ID NO: 127) (P2) was incubatedwith the SPDP-modified enzymes at an enzyme-to-DNA ratio of 1:10 in 50mM HEPES buffer (pH 7.4) for 1 h in the dark. Excess unreactedoligonucleotide was removed by ultrafiltration using Amicon 30 kD cutofffilters: washing one time with 50 mM HEPES (pH 7.4) containing 1 M NaCland three times with 50 mM HEPES (pH 7.4). The high salt concentrationin the first washing buffer helps remove DNA nonspecifically bound tothe surface of the protein due to electrostatic interactions.

The absorbance values at 260 nm and 280 nm (A₂₆₀ and A₂₈₀) were recordedto quantify the enzyme-DNA complex concentrations and the labelingratios using a Nanodrop spectrophotometer (Thermo Scientific).Extinction coefficients of DNA oligonucleotides were received fromIDT-DNA, and extinction coefficients of enzymes were obtained frompublished data.

Enzyme-DNA Cage Assembly, Purification, and Characterization

The purified DNA half-cage containing capture strands was mixed with oneof several enzyme-DNA conjugates at a 1:15 cage:enzyme ratio andannealed from 37° C. to 4° C. over 2 h in 1×TAE-Mg²⁺ buffer (containing12.5 mM Mg(OAc)2). Twenty-four single-stranded DNA linkers were mixedwith the two purified half-cages at a 5:1 linker: cage ratio to connectthe two half-cages together by incubating at room temperature for 3 h.Agarose gel electrophoresis (2%, 1×TAE-Mg21 was employed to removeexcess free enzymes (70V, 2 h). The band of the DNA cage containing theenzyme was cut from the gel and extracted using a Freeze ′N Squeezecolumn (Bio-Rad). The DNA origami concentration was quantified bymeasuring the absorbance at 260 nm (A₂₆₀) using an extinctioncoefficient of 0.109 nM⁻¹ cm⁻¹.

The DNA cage sequences are those listed in U.S. patent application Ser.No. 15/649,351, herein incorporated by reference in its entirety.

Single-Molecule Fluorescence Microscopy.

All single-molecule measurements were performed at room temperatureusing a total internal reflection fluorescence (TIRF) microscope onPEGylated fused silica microscope slides. To passivate the microscopeslides and functionalize the surface with biotin for selectiveimmobilization of nanocages, a biotin- and PEG-coated surface wasprepared by silylation with APTES, followed by incubation with a 1:10mixture of biotin-PEG-SVA 5k:mPEG-SVA 5 k as described previously. Aflow channel was constructed as described elsewhere. To prepare thesurface for enzyme or nanocage binding, a solution of 0.2 mg/mLstreptavidin in T50 buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mMEDTA) was injected in to the flow channel, incubated for 10 min, and theexcess streptavidin was flushed out thoroughly first with T50, then with1×TAE-Mg2+.

Yield estimation by TIRF colocalization: All single-moleculemeasurements were performed at room temperature using a total internalreflection fluorescence (TIRF) microscope on PEGylated fused silicamicroscope slides. To passivate the microscope slides and functionalizethe surface with biotin for selective immobilization of nanocages, abiotin- and PEG-coated surface was prepared by silylation with APTES,followed by incubation with a 1:10 mixture of biotin-PEG-SVA 5k:mPEG-SVA5k as described previously 3. A flow channel was constructed asdescribed elsewhere 3. To prepare the surface for enzyme or nanocagebinding, a solution of 0.2 mg/mL streptavidin in TSO buffer (50 mMTris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA) was injected in to the flowchannel, incubated for 10 min, and the excess streptavidin was flushedout thoroughly first with TSO, then with 1×TAE-Mg.

The right half of the DNA origami cage was labeled with Cy5 dye insidethe cavity, via hybridization of Cy5-labeled DNA to complementaryhandles incorporated into the structure. Each of the ssDNA conjugatedenzymes (HRP, GOx, G6pD, LDH, MDH and β-Gal) was covalently labeled withCy3 as described in section 3 (Cy3-Enzyme-5′-TTTTTCCCTCCCTCC, SEQ ID NO:128), and then linked to the left half of the DNA origami cage viahybridization with complementary handles. Because Cy3 was directlylabeled onto the enzyme surface, any observed Cy3 signal of theimmobilized DNA nanocages came from the encapsulated enzymes. Linkerstrands were added to a 1:1 mixture of the two half-cages to encapsulatethe enzymes in a full-cage. To capture DNA-modified enzymes in theabsence of nanocage (as control) the microscope slide was incubated with10-20 nM biotin-modified complementary DNA oligonucleotide(5′-biotin-TTTTTGGAGGGAGGG, SEQ ID NO: 129) for 3 min, followed by 10min incubation with 20-50 μM enzyme sample in 1×TAE-Mg buffer. Excessenzyme was flushed out with 400 uL buffer (channel volume 30 μL). Forthe nanocage experiments, the samples were diluted to 20-50 μM in1×TAE-Mg and immobilized on the streptavidin-coated PEG surface for 1min, and the excess sample was flushed out with 400 μL of 1×TAE-Mg. TheDNA-modified enzymes were imaged with illumination at 532 nm (15 W/cm2),and the nanocage-encapsulated enzymes were imaged with simultaneousillumination at both 532 nm (15 W/cm2) and 640 nm (40 W/cm2) asdescribed. Particle-finding and colocalization analysis were performedusing custom-written scripts in IDL and MATLAB, using a threshold of 150counts per frame for particle identification (typical particles showed500-1,000 counts per frame in each detection channel). The enzymeencapsulation yield, defined as the fraction of assembled nanocagescontaining enzyme(s), was estimated by dividing Ncaloc by the totalnumber of particles containing a right half-cage, Nright.

Estimation of enzyme copy number per nanocage: The number of enzymecopies per nanocage (Nenz) was determined by single-moleculephotobleaching (SMPB). First, the number of Cy3 photobleaching steps wasdetermined separately for unencapsulated as well as half-cage andfull-cage-encapsulated enzymes. For this, the donor channel data of allsingle molecules were idealized in QuB (http://www.qub.buffalo.edu)using a six-state model. The histogram of the photobleaching steps wasthen acquired using a custom-written MATLAB script. Finally, the numberof enzyme molecules per cage was estimated by dividing the mean numberof Cy3 photobleaching steps of the full-cage (μcy3_Encap) by the meannumber of Cy3 photobleaching steps for the unencapsulated enzyme(μcy3_Unencap). Results are summarized in Table 4.

Single-Molecule Enzymology

Single-molecule enzyme activity assay: Prior to single-molecule activitymeasurement, streptavidin-modified slides were incubated for 2 min withneutravidin-coated fluorescent beads (Invitrogen, 0.04 μm diameter,excitation/emission; 550/605 nm) at 106-fold dilution and the excessflushed out with 1×TBS buffer. These beads (5-8 per field of view) wereused as fiducial markers to correct for drift of the microscope stageand/or slide. Following complete photobleaching of Cy3 in a field ofview, the activity of single unencapsulated or nanocage-encapsulatedenzyme molecules was imaged on the same field of view. During analysisof the movies, the coordinates of the initial photobleaching movie wereregistered with those of subsequent movies using the fiducial markers(visible throughout all sequential movies) in a custom-written MATLABscript. This approach allowed us to infer the locations (x- andy-coordinates) of all individual enzymes/nanocages in the field of vieweven after bleaching Cy3, and to monitor enzyme turnovers (resorufinformation) at these specific coordinates.

To image enzyme activity, 300 μL of substrate solution in 1×TBS buffer(pH 7.5, 1 mM Mg2+, and 10% (w/v) PEG8000) was injected into the flowchannel. Movies were recorded for 5 min (9,091 frames) at 35 ms frameexposure time immediately after injecting the substrate solution. Incase of G6pDH, the activity was measured in the same field of view underidentical laser illumination and microscope settings, with or withoutglucose-6-phosphate (G6p). Enzyme activity for β-Gal was measuredsimilarly using a 500 nM solution of resorufin β-D-galactopyranoside(RBG) as substrate, which is hydrolyzed by β-Gal into fluorescentresorufin. Fluorescence fluctuations over time were measured forunencapsulated enzyme as well as half- and full-cage-encapsulatedenzyme, and the fluorescence time traces were analyzed for intensityspikes using custom-written MATLAB script. The script allowed us tomeasure the background intensity of single-molecule traces and set athreshold (mean+8 standard deviations) to subtract from the rawintensity. Since we often observed one or two spikes above thisintensity threshold in the control experiments, only those moleculeswith 2:4 spikes were counted as active molecules and considered forburst analysis. Due to the low concentration of resazurin, the criteriawe used to determine the fraction of active molecules might haveexcluded some molecules that are not highly active.

Burst analysis: Burst analysis was carried out using a modified RankSurprise (RS) method6 recently utilized to analyze the binding offluorescent DNA probes to a riboswitch. Briefly, Interspike Intervals(ISIs) were determined by calculating the time in between individualfluorescent spikes for each molecule. The RS method was used todemarcate the start and end points of bursts after collecting ISIs forall molecules. Only intensity spikes characterized by an ISIs of greaterthan 3 seconds were considered part of a burst; any other intensityspikes are counted as non-bursts.

Comparing bulk and single-molecule enzyme activity: Unlike oursingle-molecule assay, the bulk measurement of enzyme activity cannotexplicitly determine the fraction of active enzyme molecules present inthe solution (it is well known that a fraction of enzyme molecules losestheir activity during oligonucleotide conjugation, buffer exchange andthe purification process). However, the observed bulk activity iscontributed not only by enzyme turnover rate but also by the fraction ofenzyme molecules that are still active. Both of these contributingfactors need to be accounted for to directly compare the single-moleculeenzyme activity with the bulk measurements. Therefore, in thesingle-molecule experiment, the overall activity of free, half-cage andfull-cage enzymes were calculated by multiplying the turnover rate withthe fraction of active molecules for the given sample.

Bulk Solution Enzyme Assay.

A 96-well-plate reader was used to monitor enzyme activity throughabsorbance changes of the samples. The enzyme samples and substrateswere loaded in the wells of the 96-well plate with a final concentrationof caged enzymes 0.5 nM in 1×TBS (Tris buffered saline with 1 mM MgCl2,pH 7.5) for most assays.

Enzymes and Substrates:

Glucose-6-phosphate dehydrogenase (G6pDH, Leuconostoc mesenteroides),malic dehydrogenase (MDH, porcine heart), lactate dehydrogenase (LDH,rabbit muscle), glucose oxidase (GOx, Aspergillus niger), horseradishperoxidase (HRP) and P-galactosidase (β-Gal, E. coli) were purchasedfrom Sigma (St. Louis, Mo.). Pyruvate, oxaloacetate (OAA), glucose6-phosphate (G6P), glucose, resorufin 3-D-glucopyranoside (RBG),β-nicotinamide adenine dinucleotide (NAD), resazurin (RESA) andphenazine methosulfate (PMS) were obtained from Sigma-Aldrich. ABTS(2,2′-Azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt)was purchased from Pierce (Rockford, Ill.), polyphosphate (100) isordered from Kerafast.

DNA Strands:

Single-stranded MI3mp18 DNA was purchased from New England Biolabs.Staple strand oligonucleotides were obtained from Integrated DNATechnologies (IDT) on 96-well plates and used without furtherpurification. Thiol-modified DNA oligonucleotides were also purchasedfrom IDT, and were purified by denaturing PAGE before use.

Reagents:

N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) andtris(2-carboxyethyl)phosphine (TCEP) were obtained from Pierce. Dimethylsulfoxide (DMSO) was purchased from Sigma.

Buffers:

Phosphate buffered saline (PBS), HEPES sodium salt, Tris buffered saline(TBS), Tris base, acetic acid, EDTA, and magnesium acetate werepurchased from Sigma. 1×TAE/Mg2+ buffer (pH 8.0) is prepared by 40 mMTris, 20 mM acetic acid, 2 mM EDTA and 12.5 mM magnesium acetate.

Dye-Labeling Reagents:

NHS-Cy3, Cy5 amine reactive dyes were purchased from GE Healthcare LifeSciences. NHS-AlexaFluor®555 and AlexaFluor®647 amine reactive dyes wereobtained from Life Technologies.

Amicon centrifugal filters were purchased from Millipore.

PEG 8000 was purchased from Promega.

Surface PEGylating Reagents:

APTES (3-Aminopropyl)triethoxysilane was purchased from Sigma-Aldrich.mPEG-SV A 5 k and biotin-PEG-SY A 5 k were obtained from Laysan Bio,Inc.

Tem Imaging:

TEM grids (400 mesh, copper grid coated with ultrathin carbon, TedPella) were glow discharged (Emitech Kl OOX). 2 μ1 concentrated sampleswere deposited onto the grids for 1 min, washed with 10 μ1 DI water for5 sec, stained with 10 μ11% uranyl formate twice (2 sec for the firsttime and 15 sec for the second time), and imaged using Philips CMI2transmission electron microscope.

Enzyme Activity Assay:

A 96-well-plate reader was used to monitor enzyme activity throughabsorbance changes of the samples. The enzyme samples and substrateswere loaded in the wells of the 96-well plate with a final concentrationof caged enzymes of 0.5 nM in 1×TBS (Tris buffered saline with 1 mMMgCl2, pH 7.5) for most assays. The DNA cage concentration wasdetermined by the A260 value as described above. For a typical GOx andHRP assay, 1 mM Glucose and 2 mM ABTS was used as substrate and enzymeactivity was measured by monitoring the increase in absorbance at 410 nm(ABTs-1). For a typical G6pDH assay, 1 mM G6P and 1 mM NAD+ were used assubstrates, and enzyme activity was measured by monitoring the increasedabsorbance at 340 nm due to the reduction of NAD+ to NADH. For a typicalLDH assay, 2 mM pyruvate and 1 mM NADH were used as substrates, andenzyme activity was measured by monitoring the decreased absorbance at340 nm due to the oxidation of NADH to NAD+. For a typical MDH assay, 2mM OAA and 1 mM NADH were used as substrates, and enzyme activity wasmeasured by monitoring the decrease in absorbance at 340 nm. For atypical 3-Gal assay, 100 μM RBG was used as substrate and enzymeactivity was measured by monitoring fluorescence intensity, withexcitation at 532 nm and emission at 590 nm.

Trypsin Assay:

Enzyme activity was measured after incubation with or without trypsin (1μM) at 37° C. for 24 h in 1×TAE-10 mM Mg buffer (pH 8.0). Activity assayconditions: 1 mM Glucose, 1 mM ABTS, 1 nM of free GOx and HRP in pH 7.5,1×TBS buffer containing 1 mM MgCl2, and monitoring absorbance at 410 nm.In the DNA cage experiment, all conditions were the same except forincubating 1 nM DNA cage-encapsulated GOx and HRP with trypsin.

Results

Enzyme Encapsulation Strategy.

The current embodiment of the approach for enzyme encapsulation withinDNA nanocages involves two steps: 1) the attachment of an individualenzyme into an open half-cage and 2) the assembly of two half-cages intoa full (closed) nanocage. DNA half-cages were constructed by folding afull-length M13 viral DNA29 into the indicated shape based on ahoneycomb lattice using the DNA origami technique; a shape with two opensides was chosen to improve accessibility of the internal cavity tolarge proteins. Two half-cages were then linked into a full-cage byadding 24 short bridge DNA strands that hybridize with the complementaryssDNA sequences extending from the edges of either half-cage. The DNAfull-cage is 54 nm×27 nm×26 nm with designed inner cavity dimensions of20 nm×20 nm×17 nm. By design, 42 small nanopores (each 2.5 nm indiameter) were introduced on each of the top and bottom surfaces of theDNA nanocage to permit the diffusion of small molecules (e.g., enzymesubstrates) across the DNA walls.

The formation of half and full DNA nanocages was first characterizedusing transmission electron microscopy (TEM) and gel electrophoresis,which indicate a nearly 100% yield for half-cages and a more than 90%yield for full-cages. To capture target enzymes into a half-cage, apreviously reported succinimidyl 3-(2-pyridyldithio) propionate (SPDP)chemistry was used to crosslink a lysine residue on the protein surfaceto a thiol-modified oligonucleotide. Two anchor probes of complementarysequence were displayed on the bottom of the half-cage cavity to capturea DNA-modified enzyme via sequence-specific DNA hybridization.

As a demonstration of an enzyme cascade, a glucose oxidase(GOx)-attached half-cage was incubated with a horseradish peroxidase(HRP)-attached half-cage at a stoichiometric ratio of 1:1, followed bythe addition of bridge strands into solution to assemble a full DNAnanocage containing a GOx/HRP pair. The inner cavity of a full nanocageis of sufficient size to encapsulate this enzyme pair (GOx is 10 nm32and HRP 5 nm in diameter33). Unencapsulated enzyme and excess short DNAstrands were removed using agarose gel electrophoresis (AGE). Details ofthe enzyme-DNA conjugation and optimization of the assembly.

Characterization of Enzyme Encapsulation.

To verify the presence of both enzymes within a DNA nanocage, theco-localization of a Cy3-labeled GOx (green emission) and a Cy5-labeledHRP (red emission) was quantified by dual-color fluorescence gelelectrophoresis where a gel band with overlapped green and red color wasidentified. By comparison, the GOx-containing half-cage (Half[GOx])shows the presence of only Cy3 (green), whereas a HRP-half-cage(Half[HRP]) shows the presence of only Cy5 (red). In addition,negatively-stained TEM images were used to visualize DNA cages uponstoichiometrically controlled encapsulation of a single GOx or a singleGOx/HRP pair, where GOx and HRP were visible as brighter spots withinthe cage. To quantitatively analyze the yield of DNA nanocageencapsulation, two-color total internal reflection fluorescence (TIRF)microscopy34 was used to characterize the fluorescence co-localizationof a Cy3-labeled enzyme and a Cy5-labeled nanocage. Six differentenzymes were tested and characterized for encapsulation, ranging fromthe smallest HRP (44 kD)35, malic dehydrogenase (MDH, 70 kD)36,glucose-6-phosphate dehydrogenase (G6pDH, 100 kD)37, lacticdehydrogenase (LDH, 140 kD)38 and GOx (160 kD)39 to the largestP-galactosidase (β-Gal, 450 kD)40. All six enzymes were successfullyencapsulated within full DNA nanocages with high yields, ranging from64-98%. The relatively low yield of β-Gal (64%) may be due to its largesize (16 nm in diameter), which is comparable to the inner diameter ofthe nanocage (20 nm), likely resulting in steric hindrance forencapsulation. To evaluate how many copies of the same enzyme wereencapsulated per DNA nanocage, single-molecule fluorescencephotobleaching (SMPB) was used to count the number of photobleaching ofCy3 fluorophores per cage. The number of copies of each enzyme per cagewas estimated by normalizing the number of Cy3 fluorophores per DNAnanocage with the average number of Cy3 labels per free enzyme. Amajority of nanocage-encapsulated enzymes showed only one- or two-stepphotobleaching of Cy3, similar to the photobleaching of single freeenzymes. These results suggest that most nanocages (90%) contain exactlyone enzyme per cage, as expected (Table 3).

TABLE 3 Calculation of enzyme copies per DNA nanocage. The percentage ofmolecules exhibiting a given number Cy3 photobleaching steps “Cy3 Steps”for both the encapsulated and unencapsulated enzymes are provided. Themean number of enzymes per cage (N_(enz)) was calculated by taking theratio of μ_(Cy3)_Encap to μ_(Cy3)_Unencap. N is the total number ofparticles analyzed. Cy3 Steps Cy3 Steps (% molecules) (% molecules) NOne Two Three μ_(Cy3)_Encap One Two Three μ_(Cy3)_Unencap N_(enz) HRP176 86 13 1 1.15 92 8 0 1.08 1.0 G6pDH 218 87 10 3 1.16 93 7 0 1.07 1.1β-Gal 284 93 6 1 1.08 88 9 3 1.15 0.9

Conditions for the single-molecule enzyme activity assay were asfollows:

Solution Concentration 10X TBS, pH 7.5 1X Resazurin Glucose-6-phosphate50 nM (G6p) 1 nM Phenazine Methosulfate (PMS) 12.5 μM Mg²⁺ (MgCl₂) 1 mMNAD⁺ 1 mM PEG 8000 10% (w/v)

Activity Characterization of Nano-Caged Enzymes.

To evaluate the effect of DNA nanocages on enzyme activity, anencapsulated GOx/HRP pair was tested. This pair of enzymes catalyzes areaction cascade beginning with the oxidation of glucose by GOx togenerate hydrogen peroxide (H₂O₂). H₂O₂ is subsequently used by HRP tooxidize ABTS, producing a strong colorimetric signal. The overallactivity of a co-assembled GOx/HRP cage (Full[GOx/HRP]) is 8-fold higherthan that of a control enzyme pair incubated with the same cage butwithout encapsulation. Two plausible effects are hypothesized whichcould contribute to such a significant activity enhancement: 1) Theproximity effect that brings the two enzymes close together andfacilitates their substrate transfer, as described previously; and/or 2)the unique environment provided by the high charge density of DNAhelices within a nanocage.

To separate the proximity effect from the charge density effect, controlexperiments of DNA nanocages encapsulating only a single GOx or HRPenzyme are designed, which clearly do not allow for substrate channelingbetween two proximal enzymes. For example, an equimolar mixture of twoseparate nanocages encapsulating either a single GOx or a single HRP(Full[GOx]+Full[HRP]) exhibited an 4-fold increase in overall activitycompared to the unencapsulated control enzymes. Similarly, an equimolarmixture of two half-cages encapsulating either a single GOx or a singleHRP already showed an increase in overall activity by 3-fold. Sincethere was no proximity effect in the case of two enzymes encapsulatedinto two different nanocages, the local environment modified by a DNAnanocage appears to be more important for the observed activityenhancement. Similarly, a half-cage was almost as effective in activityenhancement (3-fold) as a full-cage, suggesting that enzyme access tosubstrate does not play a role in this enhancement. Interestingly, asimilar enhancement was reported previously upon conjugation of enzymesto a giant multi-branched DNA scaffold, without further explanation.

To test the generality of nanocage activity observations, the activityof six different enzymes upon encapsulation within DNA nanocages areevaluated. As shown in Table 4, five of them (GOx, HRP, G6pDH, MDH, andLDH) exhibited higher activity in nanocages than the free enzyme, withenhancements ranging from 3- to 10-fold.

TABLE 4 Enzyme kinetic data (values of K_(M) and k_(cat)) for eachindividual enzyme encapsulated inside a DNA full-cage in comparison withthe values for the free enzymes in solution. Molecular Free enzymeEncapsulated enzyme Enzyme pI weight Substrate K_(M) (μM) k_(cat) (s-1)K_(M) (μM) k_(cat) (s-1) GOx 4.2 160 kDa Glucose 6,200 ± 900  240 ± 103,000 ± 600  1,300 ± 50   HRP 8.8  44 kDa H₂O₂  2.3 ± 0.5 32 ± 1  4.3 ±0.6 290 ± 5  ABTS 2,600 ± 400  59 ± 5 2,500 ± 200  560 ± 20 G6pDH 4.3100 kDa Glucose-6-phosphate 220 ± 20 130 ± 3  310 ± 30 460 ± 10 NAD+ 510± 50 100 ± 3  590 ± 40 480 ± 10 MDH 10.0  70 kDa NADH 180 ± 50 51 ± 5270 ± 50 460 ± 30 LDH 5.0 140 kDa NADH  7.2 ± 1.3 46 ± 2 17.0 ± 1.5 190± 5  β-Gal 4.1 465 kDa RBG  58.7 ± 16.0  8.5 ± 0.6*  95.5 ± 18.9  1.6 ±0.1* ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); GOx,glucose oxidase; HRP, horseradish peroxidase; LDH, lactic aciddehydrogenase; MDH, malic dehydrogenase; pI isoelectric point. The pIvalues of the enzymes were obtained from brenda-enzymes.org *k_(cat)values for β-Gal groups were not calibrated

Detailed kinetic analyses show that the KM (the Michaelis-Mentenconstant) varies little between encapsulated and free enzyme for mostsubstrates (ranging from 0.5 to 2.4-fold of the free enzyme), suggestingthat the porous DNA cages do not substantially hinder diffusion ofsmall-molecule substrates. In contrast, a large increase in turnovernumber (kcat) was observed for these five enzymes (ranging from 3.5- to9.6-fold of the free enzyme), suggesting an inherently higher catalyticactivity of the proteins. An inverse correlation was observed betweenenhanced turnover and size of the encapsulated enzyme. That is, thesmaller HRP (44 kD) and MDH (70 kD) exhibited relatively large increasesin turnover number of 9.6±0.4 and 9.0±0.7 fold, respectively, whereasthe larger enzymes G6pDH, LDH, and GOx exhibited smaller enhancements of4.7±0.1 fold, 4.1±0.1 fold, and 5.4±0.2 fold, respectively. Nocorrelation was observed between enhancement and isoelectric point (pI),despite the wide range of pI values for these enzymes (ranging from 4.2to 10.0).

In contrast to these five enzymes, 3-Gal is strongly inhibited uponencapsulation, possibly due to its large size (16 nm in diameter) thatis comparable to the inner cavity diameter (20 nm) of the DNA nanocage.Alternatively, the 3-Gal orientation may be unfavorable and blockbinding of substrate to the active site. Notably, in a controlexperiment polyphosphate inhibited the activity of β-Gal, suggestingthat the local high density of backbone phosphates of the DNA nanocagemight be responsible for the decrease in activity of 3-Gal. The DNAcages retained their structural integrity during the enzymaticreactions.

To gain more detailed mechanistic insight into the enhancement ofcatalytic turnover, a novel single-molecule fluorescence assay tocharacterize the activity of individual enzymes with and withoutencapsulation was applied. TIRF microscopy is used to record therepetitive turnover of substrates by individual G6pDH enzymes over time;coupling with a PMS/resazurin reaction allowed us to detect stochasticfluctuations of enzyme turnover rates via transient spikes in intensityfrom the generation of the fluorescent product resorufin. Suchfluctuations have been observed for various enzymes before and arethought to be induced by the conformational switching between more andless active sub-states.

Compared to a control without substrate, more frequent fluorescentspikes were observed with the addition of glucose-6-phosphate substrate.The average spike frequency was increased from 0.016±0.001 s−1 forunencapsulated enzymes, to 0.019±0.001 s−1 for the half-cage and0.026±0.002 s−1 for the full-cage. Further analysis suggested that thefraction of active enzyme molecules was increased from 20.3% forunencapsulated enzymes to 26.6% for the half-cage and 30.5% for thefull-cage. Taken together, the 1.6-fold higher spike frequency and the1.5-fold increase in the fraction of active enzymes yield a 2.5-foldincrease in G6pDH activity for the encapsulated compared to theunencapsulated enzyme, comparable to the 4-fold enhancement observed inthe bulk assay. Conversely, a similar analysis of β-Gal activity showeda 3-fold lower activity of the full-cage enzyme (2.3±0.5 fold lower inspike frequency compared to free enzyme whereas the fractions of activeenzymes (65%) were similar) compared to unencapsulated enzyme, alsoconsistent with the bulk measurement.

The activity enhancement for DNA cage-encapsulated enzymes is consistentwith recent reports of enhanced enzyme activity upon attachment to along double-stranded DNA molecule (DNA), a 2D rectangular DNA origami,or a DNA scaffold that bound to enzyme substrates, and further suggeststhat it may be a widespread effect of enzyme-DNA interactions. Severalmechanisms have been previously proposed to explain these observedenhancements, including micro-environment composed of giant and orderedDNA molecules, molecular crowding and the substrates affinity to DNAscaffolds. We further suggested that the negatively charged phosphatebackbones of DNA might also contribute to the activity enhancement. DNAis a negatively charged biopolymer due to its closely spaced backbonephosphates (leading to a linear negative charge density of 0.6 e/A).Thus, upon encapsulation within a DNA nanocage, an enzyme is exposed toan environment full of negative charges that may resemble the relativeabundance of polyanionic molecules and surfaces (including RNA andphospholipid membranes) within the cell. Phosphate is a knownkosmotropic anion that increases the extent of hydrogen-bonded waterstructures (termed high-density or structured water). A DNA nanocage isthus expected to attract a strongly bound hydration layer ofhydrogen-bonded water molecules inside its cavity. Multiple studies havedescribed that proteins are more stable and active in a highly ordered,hydrogen-bonded water environment, possibly due to stabilization of thehydrophobic interactions of a folded protein through an increase in thesolvent entropy penalty upon unfolding.

Consistent with this model, polyphosphate has been shown to act as ageneric chaperone stabilizing a variety of enzymes. To further testwhether this mechanism is at work in our nanocages, we titrated theconcentration of NaCl (known to consist of chaotropic ions) for thepurpose of interrupting hydrogen-bonded water molecules. Consistent withour hypothesis, the activity of encapsulated enzymes significantlydecreased with increasing NaCl concentration (reduced to 25% activitywith 1 M NaCl. A high concentration of Na+ can shield the negativecharge on the DNA surface, thus disrupting the surface-bound hydrationlayer. As a control, we observed that the bulky kosmotropic cation,triethylammonium, had a much less pronounced effect on enzymaticactivity. This model also allowed us to rationalize why we observedsmaller enzymes to be more activated than larger enzymes: namely,because their higher surface-to-volume ratio predicts a stronger impactof the hydration layer.

To further test this model, we investigated the effect of DNA helixdensity on the encapsulated enzyme activity. Three nanocages weredesigned with walls that systematically increase the density of DNAhelices, including: 1) a single-layer honeycomb pattern (SH) with 2-3 nmpores between helices; 2) a single-layer square pattern (SS) withsmaller 0.5-1 nm pores between helices, and 3) a double-layer squarepattern (DS). The helix density at the top and bottom surfaces thusincreased from 0.12 helices per nm2 for SH to 0.16 helices per nm2 forthe SS and DS designs. The kcat of G6pDH encapsulated in the SH-cage was4.7-fold higher than that of the free enzyme. As the density of DNAhelices was increased, the kcat of encapsulated G6pDH raised to 6-foldfor the SS-cage and 8-fold for the DS-cage compared to the free enzymecontrol. A slight increase in KM values was also observed from theSH-cage to the SS- and DS-cages, possibly due to a decrease in substratediffusion through the DNA walls of these more tightly packed structures.For example, the KM value of G6pDH increased from 411 M in the SH-cageto 436 M in the SS-cage and 527 M in the DS-cage. Additional studiesshowed that activities of attached enzymes were enhanced by increasingthe helix packing density for various 1D, 2D and 3D DNA scaffolds. Theseobservations suggest that encapsulated enzymes exhibit higher activitywithin densely packed DNA cages, consistent with our model that thehighly ordered, hydrogen-bonded water environment near closely spacedphosphate groups are responsible for this effect.

Nanocaged enzymes are protected from proteolysis. Self-assembled DNAnanostructures previously were found to be more resistant againstnuclease degradation than single- or double-stranded DNA molecules.Similarly, DNA nanocages protect encapsulated enzymes from deactivationand aggregation under challenging biological conditions. EncapsulatedGOx/HRP was highly resistant to digestion by trypsin, and retained morethan 95% of its initial activity after incubation with trypsin for 24 h.A time-course experiment was also performed to demonstrate the stabilityof caged enzymes against Trypsin digestion. In contrast, free GOx/HRPonly retained 50% of its initial activity after a similar incubationwith trypsin. This result demonstrated the potential utility of DNAnanocages for protecting encapsulated proteins from biologicaldegradation.

Example 12

Activity of Double Nanostructure Comprising DNA Cage and RNA Nanorobot.

The experiments in Example 11 are performed using a nanostructurecomprising a DNA nanocage linked to an RNA nanorobot structure asdisclosed herein. The results from DNA cage activity test in the doublenanostructure demonstrate that encapsulated protein, in the DNA cagecomprising the double nanostructure exhibits activity as when it is insingle DNA cage structure, as shown in Example 11, above.

In some embodiments the protein in any DNA cage moiety of this inventionis a metabolic enzyme, protease and/or a therapeutic agent as describedherein.

The experiments also show that localized protein activity of the DNAcaged protein is increased by the protective, and activity enhancingeffects of the DNA cage and by the cell-specific, localizing activity ofthe RNA robot moiety.

All DNA and RNA sequences presented herein are oriented 5′->3′, unlessnoted otherwise.

Although the foregoing specification and examples fully disclose andenable certain embodiments, they are not intended to limit the scope,which is defined by the claims appended hereto.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification certainembodiments have been described, and many details have been set forthfor purposes of illustration, it will be apparent to those skilled inthe art that additional embodiments and certain details described hereinmay be varied considerably without departing from basic principles.

The use of the terms “a” and “an” and “the” and similar referents are tobe construed to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context.

The terms “comprising,” “having,” “including,” and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the technologyand does not pose a limitation on the scope of the technology unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe technology.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. It is also noted that in this disclosure and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like; e.g., they can mean “includes”, “included”,“including”, and the like; and that terms such as “consistingessentially of” and “consists essentially of” have the meaning ascribedto them in U.S. Patent law, e.g., they allow for elements not explicitlyrecited, but exclude elements that are found in the prior art or thataffect a basic or novel characteristic of the embodiment.

Embodiments are described herein, including the best mode known to theinventors. Variations of those embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the embodiments to bepracticed otherwise than as specifically described herein. Accordingly,this technology includes all modifications and equivalents of thesubject matter recited in the claims appended hereto as permitted byapplicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed byembodiments unless otherwise indicated herein or otherwise clearlycontradicted by context.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

We claim:
 1. An RNA nanostructure robot having the sequence of(R₃)_(n)—NR₁-L-NR₂—(R₄)_(m), wherein: NR₁ and NR₂ independentlyrepresent an RNA nanostructure each comprising a nucleic acid sequencehaving at least about 90% sequence identity to SEQ ID NO:1 or SEQ ID NO:9 that self-assembles into respective first and second scaffolds; L is alinker which operably links NR₁ to NR₂; wherein R₃ and R₄ areindependently selected from a pair of fastener strands, an aptamer, acargo molecule, a capture strand, a targeting strand, and H; n is aninteger from 1 to 20; and m is an integer from 0 to
 20. 2. An RNAnanostructure robot having the sequence of (R₃)_(n)—NR₁-L-NR₂—(R₄)_(m),wherein: NR₁ and NR₂ independently represent an RNA nanostructure eachcomprising a nucleic acid sequence having at least about 90% sequenceidentity to SEQ ID NO:1 or SEQ ID NO: 9 that self-assembles intorespective first and second scaffolds; L is a linker which operablylinks NR₁ to NR₂; wherein R₃ is selected from a pair of fastenerstrands, an aptamer, a cargo molecule, a capture strand, a targetingstrand, and H; n is an integer from 1 to 20; and m is an integer from 0to 20, wherein R₄ is a targeting strand operably linked to a targetingmoiety and to NR₂, wherein the targeting moiety is an aptamer thatspecifically binds nucleolin.
 3. The RNA nanostructure robot of claim 2,wherein the aptamer that specifically binds nucleolin is the F50 AS1411aptamer having the sequence: 5′-GGTGGTGGTGGTTGTGGTGG TGGTGG-3′ (SEQ IDNO: 38).
 4. The RNA nanostructure robot of claim 1, wherein the RNAnanostructure comprises a nucleic acid sequence wherein the nucleic acidsequence has at least about 95% sequence identity to SEQ ID NO:1 or SEQID NO:
 9. 5. The RNA nanostructure robot of claim 1, wherein the RNAnanostructure comprises a nucleic acid sequence wherein the nucleic acidsequence comprises SEQ ID NO:1 or SEQ ID NO:
 9. 6. An RNA nanostructurerobot having the sequence of (R₃)_(n)—NR₁-L-NR₂—(R₄)_(m), wherein: NR₁and NR₂ independently represent an RNA nanostructure each comprising anucleic acid sequence having at least about 90% sequence identity to SEQID NO:1 or SEQ ID NO: 9 that self-assembles into respective first andsecond scaffolds; L is a linker which operably links NR₁ to NR₂; whereinR₃ and R₄ are independently selected from a pair of fastener strands, anaptamer, a cargo molecule, a capture strand, a targeting strand, and H;n is an integer from 1 to 20; m is an integer from 0 to 20, wherein NR₁or NR₂ further comprises at least one operably linked therapeutic agent,and wherein the therapeutic agent is a peptide or a polypeptide.
 7. TheRNA nanostructure robot of claim 6, wherein the polypeptide therapeuticagent is a positively-charged moiety comprising 10 lysine residues. 8.The RNA nanostructure robot of claim 6, wherein the peptide therapeuticagent is a tumor targeting peptide (TTP) or a human cancer peptide. 9.The RNA nanostructure robot of claim 6, wherein the polypeptidetherapeutic agent is calreticulin.
 10. The RNA nanostructure robot ofclaim 9, wherein the calreticulin protein engages interactions betweentumor cells and macrophages or dendritic cells for enhanced antigenpresentation and stimulation of antigen-specific T cells.
 11. The RNAnanostructure robot of claim 8, wherein the human cancer peptide is ahuman NY-ESO-1 or Muc1 peptide.
 12. The RNA nanostructure robot of claim8, wherein the TTP is CTKD-K10 having the sequence:CTKDNNLLGRFELSGGGSKKKKKKKKKK (SEQ ID NO: 3).
 13. The RNA nanostructurerobot of any of claim 1, 2, or 6, wherein the RNA nanostructure robot isa TLR3 agonist.
 14. The RNA nanostructure robot of claim 1, wherein R₃is a pair of DNA fastener strands configured to fasten the first orsecond scaffold into an origami structure.
 15. The RNA nanostructurerobot of claim 1, wherein R₄ is a pair of DNA fastener strands selectedfrom the following DNA oligonucleotide pairs: 5′-FITC-labeled F50 and3′-BHQ1-labeled Comp15; FITC-F50-48 and Comp15-48-Q; FITC-F50-73 andComp15-73-Q; FITC-F50-97 and Comp15-97-Q; FITC-F50-120 and Comp15-120-Q;FITC-F50-144 and, Comp15-144-Q; and FITC-F50-169 and Comp15-169-Q;wherein the aforementioned oligonucleotides have the followingsequences: 5′-FITC-labeled F50: (SEQ ID NO: 10)5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCG TGAATTGCG-3′;3′-BHQ1-labeled Comp15: (SEQ ID NO: 11)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; FITC-F50-48: (SEQ ID NO: 12)5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCG TGAATTGCG-3′;Comp15-48-Q: (SEQ ID NO: 13)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-73(SEQ ID NO: 14) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGGAAATCAAAA-3′; Comp15-73-Q: (SEQ ID NO: 15)5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-97:(SEQ ID NO: 16) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGAACAAACTAT-3′; Comp15-97-Q: (SEQ ID NO: 17)5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-120:(SEQ ID NO: 18) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTACAAACAGTT-3′; Comp15-120-Q: (SEQ ID NO: 19)5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-BHQ1-3′; FITC-F50-144:(SEQ ID NO: 20) 5′-FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTTCCTATTATT-3;; Comp15-144-Q: (SEQ ID NO: 21)5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-BHQ1′-3′; FITC-F50-169:(SEQ ID NO: 22) 5′FITC-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTCACCTGAAA-3′; Comp15-169-Q: (SEQ ID NO: 23)5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-BHQ1- 3′[[.]];

F50 and Comp15; F50-48 and Comp15-48; F50-73 and Comp15-73; F50-97 andComp15-97; F50-120 and Comp15-120; F50-144 and, Comp15-144; and F50-169and Comp15-169; wherein the aforementioned oligonucleotides have thefollowing sequences: F50: (SEQ ID NO: 24)GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGAATTGC G-3′; Comp15:(SEQ ID NO: 25) 5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; F50-48:(SEQ ID NO: 26) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTCTAAAGTTTTGTCGTGAATTGCG-3′; Comp15-48: (SEQ ID NO: 27)5′-GTAAAGCTTTTTTTTTTTTACAACCACCACCACC-3′; FF50-73 (SEQ ID NO: 28)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGTAGAGCTTGACGGGGAAAT CAAAA-3′; Comp15-73:(SEQ ID NO: 29) 5′-TGTAGCATTTTTTTTTTTTACAACCACCACCACC-3′; F50-97:(SEQ ID NO: 30) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCGAGAAAGGAAGGGAACAAACTAT-3′; Comp15-97: (SEQ ID NO: 31)5′-TGAGTTTCTTTTTTTTTTTACAACCACCACCACC-3′; F50-120: (SEQ ID NO: 32)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGATAGGAACCCATGTACAAA CAGTT-3′; Comp15-120:(SEQ ID NO: 33) 5′-CAAGCCCATTTTTTTTTTTTACAACCACCACCACC-3′; F50-144:(SEQ ID NO: 34) 5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCACCACCCTCATTTTCCTATTATT-3;; Comp15-144: (SEQ ID NO: 35)5′-CCGCCAGCTTTTTTTTTTTACAACCACCACCACC-3′; F50-169: (SEQ ID NO: 36)5′-GGTGGTGGTGGTTGTGGTGGTGGTGGCTACATTTTGACGCTCACC TGAAA-3′; Comp15-169:(SEQ ID NO: 37) 5′-CCCTCAGTTTTTTTTTTTTACAACCACCACCACC-3′.